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2094
Biomacromolecules 2010, 11, 2094–2102
Biodegradable Block Copolymer-Doxorubicin Conjugates via
Different Linkages: Preparation, Characterization, and In Vitro
Evaluation
Xiuli Hu, Shi Liu, Yubin Huang, Xuesi Chen, and Xiabin Jing*
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences, Changchun 130022, People’s Republic of China
Received April 27, 2010; Revised Manuscript Received June 12, 2010
Doxorubicin (Dox) was conjugated onto a biodegradable block copolymer methoxy-poly(ethylene glycol)-blockpoly(lactide-co-2,2-dihydroxymethylpropylene carbonate (mPEG-b-P(LA-co-DHP)) via a carbamate linkage and
an acid-labile hydrazone linkage, respectively. Mutifunctional mixed micelles consisting of Dox-containing
copolymer mPEG-b-P(LA-co-DHP/Dox) and folic acid-containing copolymer mPEG-b-P(LA-co-DHP/FA) were
successfully prepared by coassembling the two component copolymers. The mixed micelles had well-defined
core shell structure and their diameters were in the range of 70-100 nm. Both Dox-conjugates (via carbamate or
hydrazone linkage) showed pH-dependent release behavior, and the micelles with hydrazone linkage showed
more pH-sensitivity compared to those with carbamate linkage. The in vitro cell uptake experiment by CLSM
and flow cytometry showed preferential internalization of FA-containing micelles by human ovarian cancer cell
line SKOV-3 than that without FA. Flow cytometric analysis was conducted to reveal the enhanced cell apoptosis
caused by the FA-containing micelles. These results suggested that these micelles containing both chemotherapeutic
and targeting ligand could be a promising nanocarrier for targeting the drugs to cancer cells and releasing the
drug molecules inside the cancer cells.
Introduction
Doxorubicin (Dox) is a widely used anticancer drug in the
treatment of many types of cancers.1,2 However, systemic
administration of Dox itself elicits severe cardiac toxicity due
to the lack of ability to target cancer cells, and also shows the
multidrug resistance effect.3,4 To improve therapeutic efficacy
of doxorubicin, various drug delivery systems, such as liposomes, polymeric nanoparticles, polymer conjugates, and dendrimer conjugates, have been reported.5-8
Recently, polymeric micelles of core-shell architecture based
on amphiphilic AB diblock or ABA triblock copolymers have
received much attention due to their unique structure and
characteristics, such as improved solubility and bioavailability
of hydrophobic drugs, low uptake by the reticuloendothelial
system (RES), protecting the incorporated drug from fast
degradation, blood clearance and elimination from the body,
targeting to the tumor tissue in a passive manner via the
“enhanced permeation and retention (EPR)” effect, and so
on.9-11 Dox has been physically encapsulated into and/or
chemically conjugated to polymers to prolong circulation time
in the bloodstream as well as to increase the extent of
extravascular accumulation in the tumor region.12-14 Physical
encapsulation has certain advantages such as easy preparation
and low cost, but also some disadvantages such as limited drug
loading and difficulty in drug release rate control.
Conjugation of a drug with a polymer forms a so-called
“polymeric prodrug”.15 The “prodrug” approach provides a
powerful means of drug modification, for example, solubilization
of hydrophobic drugs, elimination of initial burst release of drug
micelles, and tuning of drug pharmacokinetics. Several prodrugs
based on doxorubicin have been synthesized and evaluated. The
* To whom correspondence should be addressed. Tel./Fax: +86-43185262775. E-mail: [email protected].
primary amino and keto groups of Dox were used for covalent
attachment of Dox onto block copolymer poly(ethylene oxide)block-poly(aspartic acid) via amide bond16,17 and hydrazone
bond18 or to terminal OH-activated poly(ethylene oxide)-blockpoly(lactide).4 Ulbrich group has intensively investigated polymerDox conjugates using water-soluble macromolecular carriers
poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) via amino
or keto groups.19,20 Recent advances for these polymer-drug
conjugates have been reviewed.21-23 According to the literature,
the anticancer drug activity and toxicity of Dox is very sensitive
to the modification sites and conjugation linkage.24 On the other
hand, most of the investigated drug conjugates are based on
nonbiodegradable polymers. Biodegradable polymers have
special superiority in biomedical applications because they can
be broken down into individual monomers, which are metabolized and removed from the body via normal metabolic
pathways. In this paper, we chose amphiphilic block copolymer
mPEG-b-P(LA-co-DHP) as the carrier. Both primary amino and
keto groups of Dox were used as the conjugation sites. The
carbamate linkage and hydrazone linkage thus formed between
drug molecule and polymer were investigated.
Due to the development of biology, some specific interactions
between antibody and antigen or between ligand and receptor
have been found and used in drug delivery to realize site-specific
and time-controlled delivery.25-27 For example, it is well-known
that folic acid (FA) receptors are overexpressed in several human
tumors, including ovarian and breast cancers, and rarely
expressed in normal tissues; therefore, folic acid is covalently
conjugated to anticancer drugs or polymer micelles to achieve
selective targeting to tumors.28,29
We further prepared folic acid conjugated polymer mPEGb-P(LA-co-DHP/FA) using the same carrier. Then multifunctional micelles containing both folic acid and Dox were prepared
by coassembling the Dox-containing polymer and FA-containing
10.1021/bm100458n
 2010 American Chemical Society
Published on Web 07/07/2010
Biodegradable Block Copolymer-Doxorubicin Conjugates
polymer. Distinct from other micellar systems, the present one
exhibited the four-in-one features, that is, biodegradability,
targeting, chemotherapy, and pH sensitivity were combined
together in one micellar system. CLSM and flow cytometry were
used to monitor the internalization of the micelle drug and free
drug. MTT studies demonstrated the cytotoxic activity of the
conjugate micelles against SKOV-3 and A549 cell lines.
Experimental Section
Materials. The pendant hydroxyl group carrying copolymer mPEGb-P(LA-co-DHP), that is, methoxy-poly(ethylene glycol)-b-poly(Llactide-co-2,2-dihydroxylmethyl-propylene carbonate), was prepared
according to the literature.30 The polymerization degree of each segment
was PEG113-b-P(LA12-co-DHP8), according to 1H NMR. Doxorubicin
in the form of a hydrochloride salt was purchased from Zhejiang Hisun
Pharmaceutical Co., Ltd. 4-Nitrophenyl chloroformate (NPC) was
obtained from Sigma. Trimethylamine (TEA), methylene chloride
(CH2Cl2), and dimethylformamide (DMF) were dried over CaH2 and
distilled before use. Folic acid was purchased from Beijing Aoboxing
Universeen Biotech Co., Ltd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma. Trypsin
and RPMI 1640 medium were purchased from Gibco BRL, U.S.A.
Fetal bovine serum (FBS) was purchased from Sijiqing Biologic, China.
All other chemicals were of analytical or chromatographic grade.
Synthesis of Doxorubicin Conjugated Block Copolymer
mPEG-b-(LA-co-DHP/Dox). Synthesis of NPC-ActiVated Copolymer.
mPEG-b-(LA-co-DHP) copolymer was first activated with NPC. Briefly,
to mPEG-b-(LA-co-DHP) block copolymer (0.8 g) dissolved in methylene
chloride (20 mL), 4-nitrophenyl chloroformate (NPC; 0.195 g) and TEA
(0.3 mL) were added dropwise at 0 °C (molar ratio of DHP/NPC/TEA is
1/1.2/4). The reaction mixture was stirred for 4 h at 0 °C, and finally, the
NPC-activated mPEG-b-P(LA-co-DHP) was obtained by precipitation in
cold diethyl ether and dried in vacuo.
Conjugation of Dox onto mPEG-b-(LA-co-DHP) Copolymer Via
Carbamate Linkage. The NPC-activated mPEG-b-P(LA-co-DHP) (0.2
g) dissolved in DMF (10 mL) was reacted with Dox (50 mg) in the
presence of TEA (57 µL) for 48 h at room temperature under nitrogen.
The product mPEG-b-P(LA-co-DHP-cbm-Dox) (abbreviated as “cbmDox” hereafter) was purified as polymeric micelles by adding doubly
distilled water to the organic phase, followed by dialyzing against water
with a cellulose membrane (cutoff Mn ) 3500) for 3 days. After dialysis,
cbm-Dox was lyophilized and stored. The Dox content was determined
to be 15% by measuring the UV absorbance of a DMSO solution of
the conjugate at 480 nm. A calibration curve was constructed using a
series of concentrations of free Dox in DMSO.
Conjugation of Hydrazine on mPEG-b-(LA-co-DHP/NPC)
Copolymer. Hydrazine monohydrate (150 µL) was slowly dropped into
NPC-activated mPEG-b-P(LA-co-DHP) copolymer (0.89 g) dissolved
in methylene chloride (10 mL; molar ratio of activated hydroxyl groups/
hydrazine: 1/10). The reaction was carried out for 2 h at room
temperature. By precipitation in diethyl ether, the resulting hydrazine
conjugated mPEG-b-P(LA-co-DHP) copolymer was obtained.
Conjugation of Dox onto mPEG-b-(LA-co-DHP) Copolymer Via
Hydrazone Linkage. The hydrazone conjugated mPEG-b-P(LA-coDHP) copolymer (0.68 g) dissolved in dimethylformamide (10 mL)
was further reacted with doxorubicin (168 mg) in the presence of TEA
(0.2 mL) for 48 h at room temperature under nitrogen. The Dox
conjugate mPEG-b-P(LA-co-DHP-hz-Dox) (abbreviated as hz-Dox
hereafter) was purified as the cbm-Dox in the previous section
(conjugation of Dox onto mPEG-b-(LA-co-DHP) copolymer via
carbamate linkage). The doxorubicin content was determined to be 18%.
Conjugation of Folic Acid onto mPEG-b-(LA-co-DHP)
Copolymer. mPEG-b-(LA-co-DHP) copolymer (1.0 g) was first dissolved in 10 mL of anhydrous DMSO; then folic acid (400 mg, 0.9
mmol), DCC (185.7 mg, 0.9 mmol), and DMAP (110 mg, 0.9 mmol)
were added under stirring. The reaction mixture was stirred for 24 h at
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room temperature. Dicyclohexylurea (DCU) formed was filtered out
and the solution was dialyzed against DMSO with a cellulose membrane
(cutoff Mn ) 3500) to remove unreacted FA. Finally the product mPEGb-P(LA-co-DHP/FA) was obtained by precipitation in cold diethyl ether
and dried in vacuo.
The FA content was determined to be 20% by measuring the UV
absorbance of a DMSO solution of the conjugate at 288 nm. A
calibration curve was constructed using different concentrations of FA
in DMSO.
Physicochemical Characterization of Dox-Conjugates. 1H NMR
spectra were measured in CDCl3 or DMSO-d6 at room temperature
(20 ( 1 °C) by an AV-300 NMR spectrometer from Bruker. Gel
permeation chromatography (GPC) measurements were conducted with
a Waters 410 GPC with CHCl3 as eluent (flow rate: 1 mL/min) at 35 °C.
The molecular weights were calibrated against polystyrene (PS)
standards.
Measurement of the Micellar Size. Size distribution of the micelles
was determined by dynamic light scattering (DLS) with a vertically
polarized He-Ne laser (DAWN EOS, Wyatt Technology, U.S.A.). The
scattering angle was fixed at 90° and the measurement was carried out
at 25 °C.
TEM ObserVation. The morphology of the micelles was measured
by transmission electron microscopy (TEM) performed on a JEOL JEM1011 electron microscope operating at an acceleration voltage of 100
kV. To prepare specimens for TEM, a drop of micelle solution (1 mg/
mL) was deposited onto a copper grid with a carbon coating. The
specimens were air-dried, sputter-coated with a layer of gold, and
measured at room temperature.
In Vitro Doxorubicin Release Experiment. Freeze-dried micelle
samples (cbm-Dox and hz-Dox, 20 mg each) with either P(LA-coDHP-cbm-Dox) cores or P(LA-co-DHP-hz-Dox) cores were resuspended in acetate buffer (pH 5.0, pH 6.0) and phosphate buffered saline
(PBS, pH 7.4) solutions (5 mL), respectively, sealed in a dialysis bag
(Mw cutoff: 3.5 kDa) and incubated in the release medium (25 mL) at
37 °C under oscillation at 90 r/min. At selected time intervals, buffer
solution outside the dialysis bag was removed for UV-vis analysis
and replaced with fresh buffer solution. The released amount of Dox
was determined from the absorbance at 488 nm with the help of a
calibration curve of Dox in the same buffer. Then the accumulative
weight and relative percentage of the released Dox were calculated as
a function of incubation time.
Cell Lines. Two cell lines, including SKOV-3 (human ovarian cancer
cell line) and A549 (human lung adenocarcinoma cell line), were chosen
for cell tests. SKOV-3 cells were purchased from the Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences,
Shanghai, China, and grown in RPMI 1640 (Life Technologies,
Gaithersburg, MD) containing 10% fetal bovine serum (Life Technologies), 0.03% L-glutamine, 100 units/mL penicillin, and 100 µg/mL
streptomycin in 5% CO2 at 37 °C. A549 cells were kindly supplied by
the Medical Department of Jilin University in China and were first
cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO)
supplemented with 10% heat-inactivated fetal bovine serum (FBS,
GIBCO), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma),
and the culture medium was replaced once very day.
In Vitro Cytotoxicity. The cytotoxicity test was used to evaluate
in vitro antitumor activity of the Dox-conjugates. The folic acid receptor
positive SKOV-3 cells [FR(+)] and folic acid receptor negative A549
cells [FR(-)] were used for experiment. The cytotoxicity of the Doxconjugates on tumor cells was measured by MTT assay using Dox · HCl
solution as a control. Briefly, SKOV-3 (or A549) cells harvested in a
logarithmic growth phase were seeded in 96-well plates at a density of
105 cells/well and incubated in RPMI 1640 (DMEM for A549) for 24 h.
The medium was then replaced by the conjugate micelles or free Dox
at various Dox concentrations from 0.01 to 10 µg/mL. At the designated
time intervals (48 and 72 h), 20 µL of MTT solution in PBS with the
concentration of 5 mg/mL was added and the plate was incubated for
another 4 h at 37 °C. After that, the medium containing MTT was
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Hu et al.
Scheme 1. (A) Chemical Structure of Copolymer mPEG-b-P(LA-co-DHP); (B) Synthetic Route of mPEG-b-(LA-co-DHP/Dox) Conjugate via
Carbamate Linkage and (C) Hydrazone Linkage; (D) Synthetic Route of mPEG-b-(LA-co-DHP/FA); and (E) Concept of Micelles Forming
from mPEG-b-(LA-co-DHP/Dox)
removed and 150 µL of DMSO was added to each well to dissolve the
MTT formazan crystals. Finally, the plates were shaken for 10 min,
and the absorbance of formazan product was measured at 492 nm by
a microplate reader.
Cellular Uptake. Cellular uptake by SKOV-3 human ovarian cancer
cells was examined using flow cytometry and confocal laser scanning
microscope (CLSM), respectively. For flow cytometry studies, approximately 106 SKOV-3 cells were seeded in 6-well culture plates
and grown overnight. The cells were then treated with free Dox and
Dox-conjugated micelles (equivalent Dox concentration: 10 µg/mL)
at 37 °C for 0.5 and 2 h, respectively. After incubation, drug solutions
were removed and the cells were washed three times with 0.1 M PBS
buffer (pH 7.4). A total of 1 mL of 0.25% trypsin (GIBCO) solution
was added thereafter, and the cells were detached from cell culture by
incubation at 37 °C for 1.0 min. A single cell suspension was prepared
by filtration through a 300 mesh filter. Finally, the cells suspended in
200 µL of PBS were subjected to flow cytometry analysis using a
Becton Dickinson FACSCalibur cytometer equipped with an argon laser
(488 nm) and emission filter for 570 nm (Cytomics FC 500, BeckmannCoulter). Data were evaluated with the program WinMDI version.
For CLSM studies, SKOV-3 cells were seeded in 12-well culture
plates (a clean coverslip was put in each well) at a density of 5 × 104
cells/well and allowed to adhere for 24 h. mPEG-b-P(LA-co-DHP/Dox)
micelles or free Dox (Dox concentration: 10 µg/mL) in culture media
were added. After incubation 2 or 24 h at 37 °C, the supernatant was
carefully removed and the cells were washed three times with ice-cold
PBS. Subsequently, the cells were fixed with 800 µL of 4% formaldehyde in each well for 10 min at room temperature and washed twice
with ice-cold PBS again. Samples were examined by CLSM using a
Zeiss LSM 510 (Zurich, Switzerland) with a confocal plane of 300
nm. Dox was excited at 479 nm with emissions at 587 nm.
Drug-Induced Apoptosis Assessed by Flow Cytometry. SKOV-3
cells were seeded in sterile six-well plates at a density of 5 × 106 cells
per well. Dox and micelles (equivalent Dox concentration: 10 µg/mL)
were added into each well. After 24 h incubation, cells were collected,
centrifuged at 1000 rpm for 5 min and washed consecutively with cell
medium and PBS. Then cell pellets were resuspended in 1× Annexin
V binding buffer and Annexin V-FITC, and incubated on ice for 15
min. After incubation, the cell suspension was transferred to a FACS
tube containing propidium iodide and incubated on ice for 4 min. Cell
samples were kept on ice until flow cytometry analysis. The first 10000
events were acquired by CXP analysis software V2.1.
Results and Discussion
Construction and Physicochemical Properties of
Dox-Conjugated Micelles. The use of polymeric micelles for
cancer treatment was first reported in the early 1980s by
Ringsdorf and co-workers.31 In recent years, polymeric micelles
have been extensively exploited to improve conventional cancer
therapy. For targeting therapy, the coassembling method has
been proven to be a good choice, for it is easier and more
inexpensive than preparing the micelles from a block copolymer,
which contains both drug molecules and targeting moieties. In
this paper, folic acid (FA) was chosen as the targeting moiety.
Pendant hydroxyl carrying copolymer mPEG-b-P(LA-co-DHP)
was chosen as the carrier (Scheme 1A). Figure 1A gave its 1H
NMR spectra, and the polymerization degree of each segment
was calculated using the known molecular weight of PEG to
be PEG113-b-P(LA12-co-DHP8). The anticancer drug doxorubicin
was chemically conjugated to the pendant hydroxyl groups via
two different covalent bonds, an acid-labile hydrazone linkage
and a more stable carbamate linkage, by reacting with the amino
and keto groups in Dox, respectively, as shown in Scheme 1B,C.
The Dox content was determined to be 15% and 18% in the
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Figure 2. GPC traces of (A) mPEG-b-(LA-co-DHP) (Mw ) 12.9 ×
103, Mw/Mn ) 1.29), (B) cbm-Dox (Mn ) 9.6 × 103, Mw/Mn ) 1.16),
and (C) hz-Dox (Mn ) 11.2 × 103, Mw/Mn ) 1.17).
Figure 1. 1H NMR spectra of (A) mPEG-b-(LA-co-DHP) in CDCl3,
(B) Dox, (C) cbm-Dox, (D) hz-Dox, (E) P-FA, and (F) folic acid in
DMSO-d6.
polymer conjugate with carbamate linkage and hydrazone
linkage by measuring the UV absorbance of a DMSO solution
of the conjugates at 488 nm. Folic acid conjugate mPEG-bP(LA-co-DHP/FA) was similar to Dox-conjugates in block
backbones, amphiphilic nature, and comparable block lengths.
The only difference was that FA and the carrier polymers were
linked through an ester bond between the carboxyl group of
FA and the hydroxyl group of the copolymer backbone using
DCC and DMAP as the coupling agents (Scheme 1D). The
obtained PEG-b-P(LA-co-DHP/FA) was referred hereinafter as
P-FA for short. The folic acid content was measured by
absorption spectroscopy (DMSO solution, 288 nm) to be 20 wt
%. Both Dox and FA conjugates were also characterized by 1H
NMR as shown in Figure 1. 1H NMR supported that the
conjugation of Dox and folate to PEG-b-P(LA-co-DHP) was
successfully carried out. From the GPC analysis as shown in
Figure 2, the weight-average molecular weight (Mw) and the
molecular weight distribution (Mw/Mn) of mPEG-b-(LA-coDHP), cbm-Dox, and hz-Dox were determined to be 12.9 ×
103, 1.29; 9.6 × 103, 1.16; and 11.2 × 103, 1.17, respectively.
The molecular weight of mPEG-b-(LA-co-DHP) was a little
larger than that estimated from 1H NMR. This may be due to
the existence of the more pendant hydroxyl groups in mPEGb-(LA-co-DHP). The molecular size distribution of each conjugate was extremely narrow.
Because of the amphiphilic nature of the Dox-conjugates
(hz-Dox and cbm-Dox), they can self-assemble into micelles
using a solvent replacement method (Scheme 1E). The
hydrophobic P(LA-co-DHP/Dox) segment constitutes the core
of the micelles, and the hydrophilic PEG segment forms the
“stealth” shell of the micelles, which improves the stability
and circulation half-life of these drug-delivering micelles.32
Dynamic light scattering and transmission electron microscopy observation showed that the micelles had a spherical
shape and had an average diameter less than 100 nm for hzDox and cbm-Dox (Figure 3).
A multifunctional micellar drug delivery system, which
contains both drug and FA targeting moieties, was constructed
by coassembling cbm-Dox and P-FA (or hz-Dox and P-FA),
which carry the drug molecules and targeting moieties, respectively. The copolymer carriers, hz-Dox, cbm-Dox, and P-FA,
had similar amphiphilic nature, identical PEG length, and
comparable hydrophobic block length. Therefore, they were
expected to coassemble into mixed micelles in aqueous media,
not to form a mixture of micelles of individual copolymers.
After assembling, Dox was wrapped in the core part of the
micelles and got well protected because of its hydrophobic
nature. The FA resided in between core and corona of the
micelles to effectively play a role in targeting moieties, which
has been proven by our previous study.33 The mass ratio of the
two copolymers, hz-Dox and P-FA (or cbm-Dox and P-FA),
was 90 to 10. This ratio was based on the following consideration: the micelles should contain as much as possible drug
molecules and as little as possible targeting moieties if the
targeting capability is high enough.
In Vitro Doxorubicin Release. The ideal drug delivery
system for cancer targeting should hold the drug during
circulation but release the drug exclusively in the target. The
Dox release from the two types of conjugate micelles was
studied in acetate buffer (pH 5.0, pH 6.0) and phosphate buffered
saline (PBS, pH 7.4) solutions, respectively. As shown in Figure
4, there was no dramatic initial burst release for both cbm-Dox
and hz-Dox conjugates. This was because the Dox molecules
were chemically combined to the polymer chains and their
release from the micelles due to physical diffusion was
eliminated. However, these two drug conjugates were both pHsensitive. The lower the pH value, the faster the drug was
released. Take cbm-Dox as an example, its micelles showed
sustained release of Dox at pH 7.4, only 23% was released in
20 days. In the same time period, cbm-Dox micelles released
47% of Dox at pH 6.0 and 90% at pH 5.0. hz-Dox showed
even more significant pH sensitivity. Over 20 days, its micelles
released only 11% of Dox at pH 7.4, but 63% at pH 6.0. At pH
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Hu et al.
Figure 3. TEM images (A, B) of cbm-Dox micelles (A, C) and hz-Dox micelles (B, D) and their size distribution determined by DLS (C, D).
Figure 4. Release profiles of doxorubicin from polymer-drug conjugate micelles cbm-Dox (A) and hz-Dox (B).
5.0, 90% release of Dox took less than 10 days. These results
were ascribed to the pH sensitivities of chain degradation of
the carrier polymers and of breakage of the cbm- and hzlinkages. In the present study, the carrier polymers had the same
structure, PEG-b-P(LA-co-DHP). As reported in our previous
study,26 the PEG-b-P(LA-co-DHP) copolymer degraded more
rapidly at lower pH value. Therefore, both cbm-Dox and hzDox showed pH sensitivity. It was reported that amide bond is
more stable, while hydrazone is more acid sensitive in the case
of PHPMA-Dox conjugates.20,25 In the present study, both
carbamate and hydrazone linkages were employed. On the one
hand, the hz-Dox was more basic than the carbamate bond, due
to the hydrazone group and the NH2 group on the 3′ position
of Dox molecule, leading to slower degradation of PEG-b-P(LAco-DHP) backbone and slower release of Dox from hz-Dox
micelles at pH 7.4 (Figure 4B vs A); on the other hand, hzlinkage was more acid sensitive than the cbm-linkage, and
therefore, the hz-Dox micelles showed more significant pH
sensitivity (Figure 4B vs A).
Cellar Uptake. To demonstrate uptake and internalization
of the conjugate micelles, SKOV-3 human breast cancer cells
were chosen as target cells. Autofluorescence of doxorubicin
allows us to follow the internalization and intracellular
distribution of the tested conjugates by confocal laser
scanning microscopy (CLSM), as shown in Figure 5. The
first five pictures in Figure 5 showed the CLSM image of
SKOV-3 cells after 2 h incubation with (A) cbm-Dox, (B)
(P-FA + cbm-Dox), (C) hz-Dox, (D) (P-FA + hz-Dox), and
(E) the pristine Dox at the same 10 µg/mL equivalent Dox
concentration. It can be seen that there are limited numbers
of cbm-Dox micelles in the cells (Figure 5A). For (P-FA +
cbm-Dox) micelles (Figure 5B), the fluorescent intensity
improved obviously, indicating more micelles were internal-
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Figure 5. Confocal laser scanning microscopy of SKOV-3 cells (5 ×
104 cells/mL) after incubation with (A) cbm-Dox, (B) (P-FA + cbmDox), (C) hz-Dox, (D) (P-FA + hz-Dox), (E) free Dox for 2 h at 37 °C
and with (F) cbm-Dox, (G) (P-FA + cbm-Dox), and (H) free Dox for
24 h at 37 °C, washed three times with PBS, fixed in 4% formaldehyde, and analyzed by confocal microscope (Dox-equivalent concentration 10 µg/mL for all formulations).
ized into the cells. For both of them, the Dox was mainly
distributed in the cytoplasm. From Figure 5C and D, the FAtargeting induced preferential internalization was also observed in (P-FA + hz-Dox) micelles. Free Dox was mainly
distributed in the nucleus (Figure 5E). A comparison of
Figure 5C,D with Figure 5A,B indicates that Dox was
distributed in both cytoplasm and nucleus in hz-Dox micelles,
whereas Dox was distributed mainly in cytoplasm and rarely
in nucleus in cbm-Dox micelles, indicating that Dox release
had taken place in hz-Dox micelles. This may be because of
the acid-labile hydrazone linkage and the lower pH value in
the cell. We further investigated the uptake and internalization
of (F) cbm-Dox, (G) (P-FA + cbm-Dox) micelles, and (H)
free Dox (Figure 5) after incubation with SKOV-3 for 24 h.
Dox fluorescence was still mainly observed in the cytoplasm
while some Dox drugs entered the nucleus for the both cbmDox micelles. For free Dox, the cytoplasm and nucleus were
filled with the drug after 24 h incubation. The above CLSM
results can be summed up as the following three aspects: (1)
free Dox diffused into SKOV-3 cells very quickly. In contrast
to free drug, conjugate micelles were internalized into cells
more slowly; (2) free Dox was mainly distributed in the
nucleus of SKOV-3 cells. Fluorescence of cbm-Dox and (PFA + cbm-Dox) micelles was detectable mainly in cytoplasm
up to 24 h, while fluorescence of hz-Dox and (P-FA + hzDox) micelles was detectable in both nucleus and cytoplasm
after 2 h incubation, indicating much faster release of Dox
from hz-Dox micelles; (3) due to the presence of folate
moieties, the coassembled micelles, both (P-FA + hz-Dox)
and (P-FA + cbm-Dox), showed preferential internalization
compared to hz-Dox and cbm-Dox, respectively.
After SKOV-3 cells were incubated with various conjugate
micelles and free Dox, flow cytometry analysis was performed
to determine the mean fluorescence intensity in cells. Figure 6
gives the flow cytometry results after treatment with cbm-Dox,
(P-FA + cbm-Dox), hz-Dox, (P-FA + hz-Dox), and free Dox
for 0.5 and 2 h, respectively. As shown in Figure 6, although
identical Dox concentration (10 µg/mL) was used for these
formulations, the SKOV-3 cells incubated with FA-containing
micelles displayed higher Dox uptake compared to their
corresponding Dox conjugate micelles without P-FA. This result
was in agreement with those of CLSM observations. It implies
that FA is responsible for the enhanced uptake of the micelles.
Figure 6. (A) Mean fluorescence intensity on SKOV-3 cells after
treatment with Dox-conjugated micelles and free Dox at different
incubation time measured by flow cytometry: (A) cbm-Dox, (B) (PFA + cbm-Dox), (C) hz-Dox, (D) (P-FA + hz-Dox), (E) free Dox for
0.5 and 2 h.
Meanwhile, hz-Dox and its FA-containing micelles displayed
higher Dox uptake compared to corresponding cbm-Dox
micelles.
In Vitro Antitumor Activity. The A549 and SKOV-3 cell
lines were employed to investigate the cytotoxicity of cbmDox, (P-FA + cbm-Dox), hz-Dox, and (P-FA + hz-Dox)
micelles, with free Dox as a positive control and blank culture
medium without drugs as a negative control. Figure 7 shows
the cell viability after (A,C) 48 h and (B,D) 72 h culture with
various conjugated micelles and free Dox at various Dox
concentrations, respectively. It can be seen that cytotoxicities
of the tested formulations were all concentration dependent, and
the plot of cell viability versus logarithm of Dox concentration
is nearly linear. For the four different drug formulations [cbmDox, (P-FA + cbm-Dox), hz-Dox, (P-FA + hz-Dox)], as shown
in Figure 7A,B, the A549 cell viability level did not show an
obvious difference over the drug concentration range of 0.01
to 10 µg/mL and free Dox showed the best cytotoxicity after
incubation for 48 and 72 h. No preferred internalization for FA
containing micelles was observed because A549 cells were folate
receptor negative. No appreciable difference was observed
between cbm-Dox and hz-Dox. Both of them displayed less
cytotoxicity than free Dox. This was because free Dox molecules
were internalized and transported to the nucleus more quickly
than the conjugate micelles.
To demonstrate the contribution of FA-targeting to the
cytotoxicity, folic receptor positive SKOV-3 cells were used
for the MTT assay. As shown in Figure 7C,D, the plot of
cell viability versus logarithm of Dox concentration is linear
except the data point of free Dox at 1 µg/mL in Figure 7D.
Different from the A549 cell, the SKOV-3 cell viability level
exhibited difference between the test groups over the Dox
concentration range of 0.01 to 10 µg/mL. The order was (PFA + hz-Dox) < pristine Dox < hz-Dox < (P-FA + cbmDox) < cbm-Dox. Based on the linear relationship, the IC50
of each drug sample can be calculated and the corresponding
IC50 was 0.27, 0.49, 1.95, 7.38, and 25.3 (estimated) µg/mL.
In Figure 7D, this order changed to (P-FA + hz-Dox) < (PFA + cbm-Dox) < hz-Dox < cbm-Dox ≈ pristine Dox. The
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Hu et al.
Figure 7. Cell viabilities of A549 cells (A,B) and SKOV-3 cells (C,D) after 48 (A,C) and 72 h (B,D) incubation with various drug conjugate
micelles and free Dox at various equivalent Dox concentrations (mean ( SD and n ) 4).
Figure 8. Apoptotic cell populations determined by flow cytometric analysis with Annexin V-FITC and propidium iodide (PI) staining after incubating
SKOV-3 cells in RPMI1640 media, with (A) media alone; (B) cbm-Dox; (C) (P-FA + cbm-Dox); (D) hz-Dox; (E) (P-FA + hz-Dox); and (F) free
Dox. The lower-left and upper-left quadrants in each panel indicate the populations of normal cells and necrotic cells, respectively. While the
lower-right and upper-right quadrants in each panel indicate the populations of early and late apoptotic cells, respectively.
Biodegradable Block Copolymer-Doxorubicin Conjugates
corresponding IC50 was 0.31, 0.48, 0.64, 1.26, and 2.33 µg/
mL. Considering the fact that PEG-b-P(LA-co-DHP) itself
does not show appreciable cytotoxicity (data are not shown),
the following conclusions can be drawn: (1) cytotoxicity of
the drug-containing micelles is dependent on the linkage
between carrier polymer and drug molecule and on the FAmoieties in the micelles; (2) hz-linkage is more efficient than
cbm-linkage in causing cancer cell cytotoxicity; (3) (P-FA
+ hz-Dox) micelles exhibited the highest cytotoxicity against
folic acid receptor overexpressed cell line because both hzlinkage and FA-targeting were combined together in them.
These results were in agreement with the above drug release
and cell uptake data. The cytotoxicity of the Dox-containing
micelles was the final consequence of the following steps:
(1) internalization of the micelles, (2) Dox release from the
micelles, (3) Dox escape from the endosome, and (4) Dox
diffusion into the nucleus. The FA-moieties enhanced the
targeting and endocytosis of the micelles. In the acidic
environment of endosome, the hz-linkage was easy to break
down and, thus, Dox was released from hz-Dox micelles and
escaped from the endosome more efficiently. Because free
Dox can diffuse into the nucleus quickly, the last two steps
were not the determining steps. Therefore, the above dependences of cell cytotoxicity on linkage bonds and FA
moieties were observed. In the above experiment, performance of pristine Dox was special. On the one hand, because
it was internalized very effectively by cancer cells and it
diffused into the nucleus quickly, as indicated in the cell
uptake experiment, it exhibited quite good cell cytotoxicity
in a shorter time scale, as shown in Figure 7C; on the other
hand, because free Dox was consumed relatively rapidly, its
cytotoxicity decreased with time and finally became less than
the Dox-containing micelles (Figure 7D).
Drug-Induced Apoptosis. To investigate how the polymer
conjugated-Dox impacts its ability to induce apoptosis in vitro,
flow cytometry was conducted to evaluate the extent to which
samples of drug conjugated micelles or free drug induce
apoptosis in SKOV-3 cells. Cells were double stained for
viability (negative for propidium iodide (PI)) and apoptosis
(positive for Annexin V-FITC). Incubated with SKOV-3 cells
at a concentration of 10 µg/mL Dox-equivalent for 24 h, the
four micelles [cbm-Dox, (P-FA + cbm-Dox), hz-Dox, (P-FA
+ hz-Dox)] and free Dox resulted in 31.6, 58.6, 59.8, 64.3,
and 45.9% early apoptotic cells (LR, Annexin+/PI-) and 65.2,
35.5, 37.4, 31.2, and 51.6% normal cells (LL, Annexin-/PI-),
respectively (Figure 8). These results indicate that the biological
function of Dox was retained following conjugation to the
polymer. However, cbm-Dox micelles induce lower apoptosis
than free Dox. Taking into account the CLSM and in vitro drug
release result, this can be ascribed to the comparably slower
internalization and slower drug liberation from the carrier. As
compared to free Dox, (P-FA + cbm-Dox), hz-Dox, and (PFA + hz-Dox) micelles showed enhanced apoptosis, which is
likely due to the enhanced intracellular uptake associated with
FA-receptor-mediated endocytosis and the accelerated release
of the drug molecules from micelles with acid-labile hydrazone
linkage by sensing the acidic environment of the endosomal
compartments.
Conclusions
Two Dox-conjugates (i.e., hz-Dox and cbm-Dox) were
synthesized from biodegradable block copolymer mPEG-bP(LA-co-DHP). A higher loading of doxorubicin could be
achieved by using pendant hydroxyl-carrying copolymer
Biomacromolecules, Vol. 11, No. 8, 2010
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instead of traditional terminal functional copolymer. Multifunctional micelles consisting of Dox-containing copolymer
and folic acid-containing copolymer P-FA were successfully
prepared by coassembling the two component copolymers.
The drug molecule was wrapped in the core part of the
micelles and the hydrophilic PEG segments constituted the
corona of the micelles, leading to effective solubilization of
the Dox, while FA resided in between core and corona of
the micelles to effectively play a role of targeting moieties.
In vitro drug release studies showed that both Dox-conjugates
exhibited pH-dependent release behavior, and the micelles
with hydrazone linkage showed more pH-sensitivity compared to that with carbamate linkage. The in vitro cell uptake
experiment by CLSM and flow cytometry showed preferential
internalization of FA-containing micelles over that without
FA, in agreement with higher cytotoxicity against SKOV-3
cells tested by MTT. Flow cytometric analysis was conducted
to reveal the enhanced cell apoptosis caused by the FAcontaining micelles. These results suggested that these
micelles could be a promising drug delivery system.
Acknowledgment. Financial support was provided by the
National Natural Science Foundation of China (Project Nos.
20674084, 50733003), and by the Ministry of Science and
Technology of China (“973 Project”, No. 2009CB930102; “863
Project”, No. 2007AA03Z535).
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