Boronic acid shell-crosslinked dextran-b-PLA micelles for acid-responsive drug delivery

Article abstract of DOI:10.1002/mabi.201400251

Herein, 3-carboxy-5-nitrophenylboronic acid (CNPBA) shell-crosslinked micelles based on amphiphilic dextran-block-polylactide (Dex-b-PLA) are prepared and used for efficient intracellular drug deliveries. Due to the reversible pH-dependent binding with diols to form boronate esters, CNPBA modified Dex-b-PLA shows excellent pH-sensitivity. In neutral aqueous conditions, CNPBA-Dex-b-PLA forms shell-crosslinked micelles to enable DOX loading, while in acid conditions, the boronate esters hydrolyze and the micelles de-crosslink to release loaded DOX. In vitro release studies indicate that the release of the DOX cargo is minimized at physiological conditions, while there is a burst release in response to low pHs. The cell viability of CNPBA-Dex-b-PLA investigated by MTT assay was more than 90%, indicating that, as a drug delivery system, CNPBA-Dex-b-PLA has good cytocompatibility. These features suggest that the pH-responsive biodegradable CNPBA-Dex-b-PLA can efficiently load and deliver DOX into tumor cells and enhance the inhibition of cellular proliferation in vitro, providing a favorable platform as a drug delivery system for cancer therapy.

Full text of DOI:10.1002/mabi.201400251

Full Paper
Boronic Acid Shell-Crosslinked Dextran-b-PLA
Micelles for Acid-Responsive Drug Deliverya
Ziwei Zhao, Xuemei Yao, Zhe Zhang, Li Chen,* Chaoliang He, Xuesi Chen
Herein, 3-carboxy-5-nitrophenylboronic acid (CNPBA) shell-crosslinked micelles based on
amphiphilic dextran-block-polylactide (Dex-b-PLA) are prepared and used for efficient
intracellular drug deliveries. Due to the reversible pH-dependent binding with diols to form
boronate esters, CNPBA modified Dex-b-PLA shows excellent pH-sensitivity. In neutral
aqueous conditions, CNPBA-Dex-b-PLA forms shell-crosslinked micelles to enable DOX loading,
while in acid conditions, the boronate esters hydrolyze and the micelles de-crosslink to release
loaded DOX. In vitro release studies indicate that the release of the DOX cargo is minimized at
physiological conditions, while there is a burst release in response to low pHs. The cell viability
of CNPBA-Dex-b-PLA investigated by MTT assay was more than 90%, indicating that, as a drug
delivery system, CNPBA-Dex-b-PLA has good cytocompatibility. These features suggest that
the pH-responsive biodegrad-
able CNPBA-Dex-b-PLA can ef-
ficiently load and deliver DOX
into tumor cells and enhance
the inhibition of cellular pro-
liferation in vitro, providing a
favorable platform as a drug
delivery system for cancer
therapy.
1. Introduction
due to their accumulation in tumors via the passive
‘‘enhancedpermeabilityandretention(EPR)effect’’. Among
these nanocarriers, stimuli-responsive nanoparticles offer
the opportunity to selectively deliver a drug on demand to a
specific location within the human body. External or
physiological stimuli, such as pH,[7–9] temperature,[10–11]
light[12–13], reductive,[13–15] or enzymes,[16] have attracted
tremendous attention as triggers for intelligent carriers to
release drugs. These stimuli responsive carriers are noted
for their site-specific targeted release of payloads modulat-
ed by the specific microenvironments of the intracellular
space, leading to aggressive anticancer activity and
maximal chemotherapeutic efficacy with fewer side
effects.[17]
Over the past few decades, nanocarriers have emerged as
one of the most fascinating delivery vehicles for small
molecular antitumor drugs, such as doxorubicin (DOX),[1–4]
paclitaxel (PTX),[5] and docetaxel,[6] to improve therapeutic
efficiency and eliminate adverse effects on organisms
Z. Zhao, X. Yao, Z. Zhang, L. Chen
Department of Chemistry, Northeast Normal University,
Changchun 130024, P. R. China
E-mail: chenl686@nenu.edu.cn
C. He, X. Chen
Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun 130022, P. R. China
Compared with the neutral pH environment in most
human tissues, mildly acidic conditions may be found in
sites of inflammation, lysosomal compartments, or tumor
aSupporting Information is available at Wiley Online Library or from
the author.
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Scheme 1. Schematic illustration of DOX loading and release from DOX-loaded PDP-m.
tissue. Because of this change in the physiological
environment, choosing pH as an internal stimulus has
become the most common method used to prepare smart
responsive drug delivery systems. Compared with the
conventional specific tumor cell surface targeting ap-
proach, this pH-targeting approach is considered to be a
more general strategy because of the acidic tumor
microclimate in solid tumors. Hence, many acid-labile
chemical bonds, such as maleic acid amide,[18] acetal,[19]
hydrazone,[20] and orthoester,[21] have been extensively
employed to design acid-responsive drug delivery
systems.
hydrophobic phenylboronic ester functionalities to spon-
taneously form acid-responsive nanoparticles in water.[34]
Therefore, boronic ester-containing polymers with unique
reactivity and self-assembly capability have significant
potential applications as sensors for sugars and glycopro-
teins, and in self-regulated drug delivery systems and
therapeutic agents.
Polymeric micelles based on amphiphilic copolymers, as
one kind of antitumor drug nanocarrier, have received
considerable attention due to their greatly enhanced
water solubility and their prolonged circulation in blood.
However, one practical challenge with their application is
their low stability in vivo. In order to overcome this
limitation, different shell or core crosslinking approaches
have been adopted, which can not only improve the
stability of micelles, but also control drug release through
environment responsive linkages. Herein, 3-carboxy-5-
nitrophenylboronic acid (CNPBA) was chosen as a cross-
linker to prepare shell-crosslinked micelles based on Dex-b-
PLA copolymers. Dex-b-PLA copolymers were first prepared
by a click reaction and were then modified by the
crosslinker CNPBA to obtain CNPBA-Dex-b-PLA. Taking
advantage of its pH-sensitivity, DOX, as a model anticancer
drug, was loaded into the shell-crosslinked micelles, as
shown in Scheme 1. This type of pH-sensitive micelle could
rapidly respond to the acidic intracellular compartments of
carcinoma cells, and therefore holds great potential for in
vivo chemotherapeutics.
It is well known that boronic acids can reversibly bind
with diols to form boronate esters, one of the key precursors
of boronic acid derivatives, which exhibit fast responsive-
ness to both the external pH environment and competing
diols.[22–24] Under acidic conditions, boronic esters exhibit
pH-responsiveness and can be easily hydrolyzed. As a kind
of boronic acid, phenylboronic acid(PBA) anditsderivatives
can specifically interacted with mannitol and other polyols
to form phenylboronates. There are two forms of PBA
moieties at equilibrium in water: (1) the neutral and
hydrophobic form with the triangular plane structure
(pH < pKa of PBA); (2) the charged and hydrophilic form
where PBA groups have a tetrahedral structure (pH > pKa of
PBA).[25] Based on these two forms, phenylboronates show
an excellent pH-sensitive property. On the other hand, the
formation of boronate esters can further improve the
hydrophilicity of PBA-functionalized polymers, resulting in
the swelling of the matrix and the release of payloads.
Taking advantage of this reversible change, PBA has been
chosen as a promising candidate for nanocarriers and has
been immobilized in various matrices.[26–32] Zhang et al.
have reported the formation of pH-responsive polymer
nanoparticles by crosslinking dextran with poly(3-acryl-
amidophenylboronic acid).[33] Li has modified dextran with
2. Experimental Section
2.1. Materials
L-Lactide (L-LA, Purac) and D-lactide (D-LA, Purac) were recrystallized
threetimesfromethylacetateunderanargonatmosphere.Dextran
(Dex, Mn ¼ 6 kDa, Sigma), 3-bromo-1-propanol (Sigma), sodium
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azide (Sigma), stannous octoate (tin (II)-bis (2-ethylhexanoate)
(Sn(Oct)2, 95% Sigma), propargylamine (98% Sigma), sodium
cyanoborohydride (Sigma), N,N,N0,N0,N00-pentamethyldiethylene-
triamine (PMDETA, Sigma), 3-carboxy-5-nitrophenylboronic acid
(Sigma), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)
(Sigma), 4-dimethylaminopyridine (DMAP) (Sigma), and copper
bromide (Sigma) were used as received. Toluene was dried by
refluxing over Na metal under an argon atmosphere and distilled
immediatelybeforeuse. Dimethylsulfoxide (DMSO)wasdriedover
calcium hydride (CaH2) and purified by vacuum distillation with
CaH2. Acetate buffer with a concentration of 0.2 mol Lꢀ1 was
prepared by mixing acetic acid and sodium acetate at pH 5.0. Dry 3-
(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) was purchased from Sigma-Aldrich. Doxorubicin hydrochlo-
ride (DOX ꢁ HCl) was purchased from Zhejiang Hisun Pharmaceuti-
cal Co., Ltd.
weight of N3-PLA was calculated from 1H NMR and GPC
measurements.
2.4. Synthesis of a-Alkyne Dextran (Alkyne-Dex)
ThemodificationofdextranwasconductedasshowninScheme2b.
Typically,dextran(4.0 g, 0.66 ꢂ 10ꢀ3 mol)wasdissolvedin2%(w/v)
acetate buffer (pH 5.0) in a flask at 50 8C. Propargylamine (0.684 g,
9.8 ꢂ 10ꢀ3 mol, 15 equiv.) and sodium cyanoborohydride (0.628 g,
9.9 ꢂ 10ꢀ3 mol, 15 equiv.) were added under stiring. The mixture
was allowed to stir at 50 8C for 96 h. The solution was then
concentrated using a rotavapor, and dialyzed against deionized
water (MWCO 3.5 kDa) for 2 d. The product was collected by
lyophilization (2.8 g; yield: 70%).
2.5. Synthesis of Dextran-block-Polylactide (Dex-b-
PLA)
2.2. Characterization
Dex-b-PLA was synthesized by Huisgen’s 1,3-dipolar cycloaddition
(‘‘click chemistry’’) shown in Scheme 2c. A typical procedure for the
preparationisbrieflydescribedasfollows.N3-PLA(0.2 g,0.06 ꢂ 10ꢀ3
mol), alkyne-Dex (0.6 g, 0.1 ꢂ 10ꢀ3 mol) and PMDETA (21 mL,
0.1 ꢂ 10ꢀ3 mol) were dissolved in 30.0 mL of dried DMSO with
stirring for 30 min. After being degassed by three freeze-thaw
cycles, the mixture was transferred into another Schlenk flask
containing CuBr (14.4 mg, 0.1 ꢂ 10ꢀ3 mol) via a N2-purged syringe
in an oil bath at 60 8C for 72 h. After the reaction finished, the
reaction medium was dialyzed against deionized water (MWCO
7 kDa) for 4 d and the product was obtained by lyophilization (0.5 g;
yield: 63%).
1H NMR spectra were recorded on a Bruker AV 400 NMR
spectrometer in dimethyl sulfoxide-d6 (DMSO-d6) or chloroform-
d1 (CDCl3-d1). Fourier Transform infrared (FTIR) measurements
wererecordedonaBio-RadWin-IRinstrument usingthepotassium
bromide (KBr) method. Gel permeation chromatography (GPC)
analyses were performed on a Polymer Laboratories PL-GPC 50 Plus
integrated GPC system with two Jordi DVB mixed bed columns
(300 ꢂ 7.5 mm) and absolute molecular weights were obtained
using a refractive index detector coupled to a multiangle laser light
scatteringdetector, theMiniDAWNTreos(WyattTechnology, USA).
GPC analyses were carried out in HPLC-grade CHCl3 (flow rate:
1 mL minꢀ1) at 50 8C on samples of 1 mg mLꢀ1 concentration.
Dynamiclightscattering(DLS)measurementswereperformedona
WyattQELS instrument with a vertically polarized He-Ne laser
(DAWN EOS, Wyatt Technology) and 908 collecting optics. All the
samples were prepared in aqueous solution at a concentration of
0.25 mg mLꢀ1. Transmission electron microscopy (TEM) measure-
ments were performed using a JEOL JEM-1011 transmission
electron microscope with an accelerating voltage of 100 kV. A
drop of the sample solution (0.25 mg mLꢀ1) was deposited onto a
230 mesh copper grid coated with carbon and allowed to dry in air
at 25 8C before measurement.
2.6. Synthesis of CNPBA Modified Dex-b-PLA (CNPBA-
Dex-b-PLA)
The mechanism is based on the condensation reaction, as shown in
Scheme 2d. Typically, Dex-b-PLA (1.0 g, 0.11 ꢂ 10ꢀ3 mol), CNPBA
(0.17 g, 0.82 ꢂ 10ꢀ3 mol), EDC (0.31 g, 1.64 ꢂ 10ꢀ3 mol, 2 equiv.), and
DMAP (0.02 g, 0.16 ꢂ 10ꢀ3 mol, 2 equiv.) were dissolved in 15.0 mL
of DMSO with stirring for 24 h at room temperature. After the
reaction finished, the reaction medium was dialyzed against
deionized water (MWCO 7 kDa) for 4 d and the product was
obtained by lyophilization (0.81 g; yield: 69%).
2.3. Synthesis of a-Azido PLA (N3-PLA)
The introduction of the azide group to the PLA backbone was
conducted in two steps described in Scheme 2a. First, sodium azide
(0.3 g, 4.62 ꢂ 10ꢀ3 mol) was dissolved in 20.0 mL of 3-bromo-1-
propanol in a flame-dried flask and the reaction was maintained
at room temperature for 48 h. Second, an equimolar mixture of
L-lactide and D-lactide (10.0 g, 69.4 ꢂ 10ꢀ3 mol) was dissolved in
30.0 mL of anhydrous toluene in a flame-dried flask at room
temperature. Then, a-azido propanol (0.19 mL, 2.5 ꢂ 10ꢀ3 mol) and
Sn(Oct)2 (28.12 mg, 69.4 ꢂ 10ꢀ6 mol, 0.001 equiv.) were added into
the solution with stirring and the ROP reaction was maintained at
85 8C for 4 d. After precipitating the solution into excessive diethyl
ether, the crude product was further washed twice with diethyl
ether and dried under a vacuum at room temperature for 24 h to
obtain the resultant N3-PLA (8.0 g; yield: 80%). The molecular
2.7. In Vitro Drug Loading and Release
Doxorubicin (DOX) was chosen as a model drug for in vitro drug
loading and release. DOX-loaded micelles were prepared by a
simple dialysis technique. Typically, PDP-m (15 mg), DOX ꢁ HCl
(3.0 mg), and triethylamine (0.9 mg)were mixed in 2.0 mL of DMSO.
The mixture was stirred at room temperature for 24 h and then
added dropwise into 20.0 mL of PBS at pH 7.4. The DMSO was
removed by dialysis against deionized water for 24 h. The dialysis
medium was refreshed five times and the whole procedure was
performed in the dark. Then, the solution was filtered and
lyophilized. To determine the drug loading content (DLC) and drug
loading efficiency (DLE), the drug-loaded micelles were dissolved in
DMSO and analyzed by fluorescence measurements (Perkin-Elmer
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Scheme 2. Synthetic routes to CNPBA-Dex-b-PLA.
LS50B luminescence spectrometer) using a standard curve method
(lex ¼ 480 nm). The DLC and DLE of DOX-loaded micelles were
calculated according to Equation (1) and Equation (2), respectively:
3.5 kDa). The release experiment was initiated by placing the end-
sealed dialysis bag into 50 mL of PBS at 37 8C with continuous
shaking at 100 rpm. At pre-determined time intervals, 2.0 mL of
dialysate was taken out and an equal volume of fresh PBS was
replenished. The amount of released DOX was determined by
fluorescence measurements (lex ¼ 480 nm). The release experi-
ments were conducted in triplicate.
amount of drug in micelles
DLCðwt%Þ ¼
DLEðwt%Þ ¼
ꢂ 100
ð1Þ
ð2Þ
amount of drug loaded micelles
amount of drug in micelles
total amount of feeding drug
ꢂ 100
2.8. Intracellular Drug Release
In vitro drug release profiles of DOX-loaded micelles were
investigatedin PBS atpH5.5, 6.8, and 7.4, respectively. Theweighed
freeze-dried DOX-loaded micelles were suspended in 10.0 mL of
release medium and transferred into a dialysis bag (MWCO
The cellular uptake and intracellular release behaviors of DOX-
loaded micelles were assessed by confocal laser scanning
microscopy (CLSM) and flow cytometry on MCF-7 cells.
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2.8.1. Confocal Laser Scanning Microscopy (CLSM)
3. Results and Discussion
For CLSM studies, MCF-7 cells were seeded in 6-well plates at a
density of 1 ꢂ 105 cells per well in 2.0 mL of complete Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine
serum, supplemented with 50 IU mLꢀ1 penicillin and 50 IU mLꢀ1
streptomycin, and cultured for 24 h. After the culture media were
removed,thecellswereincubatedat37 8Cforanadditional3 hwith
DOX-loaded micelles at a final DOX concentration of 10.0 mg Lꢀ1 in
complete DMEM. Then, the culture medium was removed and cells
were washed with PBS three times. Thereafter, the cells were fixed
with 4% paraformaldehyde for 30 min at room temperature, and
the cell nuclei were stained with 40,6-diamidino-2-phenylindole
(DAPI,blue)for20 min. CLSMimagesofcellswereobtainedthrough
a confocal microscope (Olympus FluoView 1000).
3.1. Synthesis of CNPBA-Dex-b-PLA
Dex-b-PLA was synthesized by combining the clickable
alkyne-Dex with N3-PLA precursor via click chemistry, as
depicted in Scheme 2. Firstly, atactic-PLA was synthesized
via the ring-opening polymerization of an equimolar
mixture of -lactide and D-lactide by varying the ratio of
L
lactide to a-azido propanol. The 1H NMR spectrumof N3-PLA
in CDCl3 is shown in Figure S1 in the Supporting
Information. The peaks at 5.15–5.25 ppm and 1.5 ppm were
attributed to the characteristic signals of the PLA units.
According to the 1H NMR spectrum,[35] the molecular
weight of atactic-PLA was 3000 in accordance with that
obtainedfromgelpermeationchromatography(GPC). From
GPC, the molecular weight of atactic-PLA was 3346 with a
PDI of 1.84. In order to conduct the click reaction, an alkyne
group was introduced into dextran by reductive amination
with propargylamine in acetate buffer solution (pH 5.0).
The chemical structure of alkyne-Dex was confirmed by 1H
NMR. As shown in Figure S2 in the Supporting Information,
the complete disappearance of the anomeric proton peaks
at 6.7 and 6.3 ppm belonging to the reducing end group
quantitatively indicated the successfully synthesis of a-
alkyne dextran. Then the amphiphilic Dex-b-LA was
synthesized by a click reaction. As shown in Figure 1, the
methine protons peaks at 5.15–5.25 ppm were attributed to
the characteristic signals in PLA units and the peaks at 3–
4 ppm were assigned to the protons of the dextran. This
further indicated the successful synthesis of amphiphilic
Dex-b-PLA copolymers.
2.8.2. Flow Cytometric Analysis
MCF-7 cells were seeded in 6-well plates at 1 ꢂ 105 cells per well in
2.0 mL of completeDMEMand cultured for24 h. Thecells were then
washed with PBS and incubated at 37 8C for an additional 3 h with
DOX-loaded micelles at a final DOX concentration of 10.0 mg Lꢀ1 in
completeDMEM.Thereafter, theculturemediumwasremovedand
the cells were washed with PBS three times and treated with
trypsin. Then, 1.0 mL of PBS was added to each culture well, and the
solutionswerecentrifugedfor5 minat1000 rpm.Aftertheremoval
of supernatants, the cells were resuspended in 0.2 mL of PBS. Data
for1 ꢂ 104 gatedeventswerecollected,andanalysiswasperformed
by flow cytometry (Beckman, California, USA).
2.9. Cell Viability Assays
The relative cytotoxicities of micelles against HeLa cells and MCF-7
cells were evaluated in vitro using a standard MTT assay. The cells
were seeded in 96-well plates at 7000 cells per well in 200.0 mL of
complete DMEM and incubated at 37 8C in a 5% CO2 atmosphere for
24 h. The culture medium was then removed and micelle solutions
in complete DMEM at different concentrations (0–10 g Lꢀ1) were
added. The cells were subjected to the MTT assay after being
incubated for an additional 72 h. The absorbance of the solution
was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell
viability (%) was calculated based on Equation (3):
A
Cell viabilityð%Þ ¼ sample ꢂ 100
ð3Þ
Acontrol
where, Asample and Acontrol represent the absorbance of the sample
and control wells, respectively.
The cytotoxicities of DOX-loaded micelles against HeLa cells and
MCF-7 cells were also evaluated in vitro by a MTT assay. Similarly,
cells were seeded into 96-well plates at 7000 cells per well in
200.0 mL of complete DMEM and incubated for 24 h. After washing
cells with PBS, 180.0 mL of complete DMEM and 20.0 mL of DOX-
loaded micelle solutions in PBS were added to form culture media
with different DOX concentrations (0–10.0 mg Lꢀ1 DOX). The cells
were subjected to the MTT assay after being incubated for 24, 48,
and72 h.TheabsorbanceofthesolutionwasmeasuredonaBio-Rad
680 microplate reader at 490 nm. Cell viability (%) was also
calculated based on Equation (3).
Figure 1. 1H NMR spectrum of Dex-b-PLA in DMSO-d6.
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Table 1. Characterization of PDP-m.
Sample
Entry
Ratioa) [%] Ratiob) [%] CMCc) [mg mLꢀ1
]
DLSc) [nm] DLC [wt%] DLE [wt%]
Dex-b-PLA
PDP-0
–
5
–
0.033
0.033
0.027
0.021
59.9 ꢃ 6.2
56.4 ꢃ 5.7
44.2 ꢃ 5.3
31.1 ꢃ 4.8
8.7
9.0
51.2
53.7
56.7
61.3
CNPBA1-Dex-b-PLA PDP-1
CNPBA2-Dex-b-PLA PDP-2
CNPBA3-Dex-b-PLA PDP-3
3.7
9.5
17.3
10
20
9.3
10.2
a)Theoretical grafting ratio of CNPBA, refered to as the ratio of CNPBA to glucose residue; b)Actual grafting ratio of CNPBA by UV-vis;
c)Determined in PBS at 7.4.
CNPBA-Dex-b-PLAs were prepared by a condensation
reaction via the hydroxyl group of Dex-b-PLA and the
carboxyl group of CNPBA. FT-IR spectra are shown in
Figure S3 in the Supporting Information. The peak at 1530
cmꢀ1 belonging to the phenyl group suggested the
successful modification of Dex-b-PLA by CNPBA. The
grafting ratios of CNPBA were calculated by a UV-vis
method, as show in Figure S4 in the Supporting Informa-
tion. According to the standard curve, the actual grafting
ratio of CNPBA in PDP-m was obtained, as listed in
Table 1.
different becauseofthe disassemblyandre-assembly ofthe
crosslinked micelles.
The hydrodynamic radii (Rh) of the amphiphilic aggre-
gates were also measured by DLS, as listed in Table 1 and
Figure 2A. At pH 7.4, the average hydrodynamic diameters
(Rh) of the crosslinked micelles were 56.4 ꢃ 5.7 nm (PDP-1),
44.2 ꢃ 5.3 nm (PDP-2), and 31.1 ꢃ 4.8 nm (PDP-3), respec-
tively, which were much lower than that of uncrosslinked
micelles (PDP-0, 59.9 ꢃ 6.2 nm). Crosslinked micelles had
smaller particle diameters due to the reaction between
dextran and CNPBA. Interestingly, the shell-crosslinked
micelles led to a continuous decrease in Rh as the amount of
CNPBA (3.7ꢄ17.3%) increased. The pH-responsive behavior
of the micelles was also investigated at different pH values
using DLS measurements. As shown in Figure 2B, the Rh of
PDP-2 was smaller and the distribution was relatively
narrower at pH 7.4. However, there appeared to be a clear
bimodal distribution due to the disassembly and re-
assembly of PDP-2 micelles in the acidic environment
(pH 5.5). The same results could also be certified by
transmission electron microscopy (TEM, Figure 2C and 2D).
The smaller values from TEM observations when compared
to DLS will be due to the dehydration of the micelles in the
process of TEM sample preparation. These behaviors can be
explained by the fact that the boronate ester bond is
unstable and would hydrolyze in an acid environment,
resulting in the disassembly and re-assembly of the
micelles. All of these findings inferred that the crosslinked
micelles had a good response to low pH values.
3.2. Characterization of Shell-Crosslinked Polymer
Micelles
It is known that boronic acid can easily react with hydroxyl
groups to form boronate ester in neutral aqueous solution.
There are abundant functional hydroxyl groups along the
chain of dextran, so CNPBA-Dex-b-PLA can self-assemble to
form shell-crosslinked micelles in neutral aqueous con-
ditions. The self-assembly of CNPBA-Dex-b-PLA was char-
acterized by fluorescence spectra using Nile Red as the
fluorescence probe, according to a literature method.[36–37]
The critical micelle concentration (CMC) could be calculated
by tracking the fluorescence intensity of Nile Red as a
function of the sample concentration, as shown in Figure S5
in the Supporting Information and Table 1. At pH 7.4, PDP-3
showed a lower CMC value of 0.021 mg mLꢀ1 than that of
PDP-0 (0.033 mg mLꢀ1) because boronic acid from CNPBA
reacted with the hydroxyl group of dextran to form shell-
crosslinked micelles at lower concentrations. The CMC
values of PDP-0 and PDP-1 were very similar due to the
actual grafting ratio of CNPBA for PDP-1 being only 3.7%.
As is well known, crosslinked boronate ester bonds
undergohydrolysisinacidicconditions, whichwouldresult
in the de-crosslinking of the crosslinked micelles. This pH-
sensitivity was further verified by the change of CMC value
with pH value. For PDP-0 micelles, the CMC values were the
same at both pH 5.5 and pH 7.4, as shown in Figure S6 and
Figure S7 in the Supporting Information. However, for PDP-
3 micelles, the CMC values at pH 5.5 and pH 7.4 were quite
3.3. In Vitro DOX Loading and Triggered Release
Doxorubicin is a widely used antineoplastic agent in the
treatment of several adult and pediatric cancers, which
interacts with DNA through insertion and then inhibits the
biosynthesis of bioactive macromolecules. In the current
study, DOX was used as a model drug and loaded into the
micelles. As shown in Table 1, the DLC of PDP-0ꢄ3 were in
the range 8.7–10.2% and the DLE were in the range 51.2–
61.3%. The data indicated that the stronger crosslinked
interaction of the micelles enhanced the drug loading
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Figure 2. The Rh and size distributions of PDP-m at pH 7.4 (A); the Rh and size distributions of PDP-2 at pH 7.4 and 5.5, respectively (B); TEM
micrographs of PDP-2 in pH 7.4 (C) and pH 5.5 (D).
capacity (DLC and DLE). The in vitro release behaviors were
investigated at pH 5.3, 6.8, and 7.4. Figure 3 shows the
cumulative release percentages of DOX-loaded micelles
versus time. From this, it can be concluded that: (1) at the
same pH value, a higher ratio of CNPBA in the polymer
resultedinafasterreleaserateandagreatertotalamountof
release, because the boronate ester could improve the
hydrophilicity of CNPBA functionalized polymers which
resulted in the swelling of the matrix and the release of the
payloads; (2) up to 90% of DOX was released from DOX-
loaded PDP-3 micelles during the first 12 h in PBS at pH 5.5,
and the DOX release rate decreased with increasing pH
values 5.5 to 7.4. All this data shows that the DOX-loaded
crosslinked micelles have an excellent pH-responsive
property and great potential as in vivo intelligent drug
delivery vectors.
3.4. Intracellular DOX Release and Cellular
Proliferation Inhibition
The cellular uptake and intracellular release behaviors of
DOX-loaded micelles were followed with CLSM and flow
cytometry toward MCF-7 cells. As expected, a stronger
intracellular DOX fluorescence was observed in the cells
after incubation with DOX-loaded PDP-3 micelles for 2 h, in
contrast to those incubated with DOX-loaded PDP-0 micelle
(Figure 4AꢄB). The intracellular triggered drug release
behavior was further confirmed by flow cytometric
analysis. As shown in Figure 5, the flow cytometric
Figure 3. In vitro DOX release from DOX-loaded PDP-m in PBS
at pH 5.5, 6.8, and 7.4, respectively.
Macromol. Biosci. 2014, DOI: 10.1002/mabi.201400251
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histogram for the cells incubated with DOX-loaded PDP-3
micelles shifted clearly to the direction of higher fluores-
cence intensity when compared with the cells incubated
with DOX-loaded PDP-0 micelles. This enhanced fluores-
cence intensity in the MCF-7 cells incubated with DOX-
loaded PDP-3 micelles should attributed to the lower pH
environment, whichinduced the swellingofthematrix and
the release of the payloads.
It is necessary to evaluate the potential toxicity of
polymeric materials for drug delivery applications. Usually
MTT, WST-1, and Cell Titer can be used to evaluate the
cytotoxicity of cells. Herein, we chose MTT, the method
most frequently used in our laboratory, to study the
cytotoxicity. The in vitro cytotoxicity of PDP-m to MCF-7
and HeLa cells was evaluated by a MTT assay. As shown in
Figure 6, the viabilities of MCF-7 and HeLa cells treated with
PDP-0ꢄ3for72 hwereover80%atalltestconcentrations up
to 10.0 g Lꢀ1. These results suggested that both uncros-
slinked and crosslinked micelles had low cytotoxicity and
could be safely used as biocompatible carriers for drug
delivery.
Figure 4. Representative CLSM images of MCF-7 cells incubated
with DOX-loaded PDP-0 (A) and DOX-loaded PDP-3 (B). For each
panel, the images from left to right show differential interference
contrast (DIC) images, cell nuclei stained by DAPI (blue), DOX
fluorescence in cells (red), and overlays of the three images.
The in vitro cellular proliferation inhibitions of DOX-
loaded PDP-m against MCF-7 and HeLa cells were also
estimated by a MTT assay. In contrast to DOX-loaded PDP-0,
DOX-loaded PDP-1ꢄ3 exhibited significantly lower growth
inhibition efficiency to MCF-7 and HeLa cells (shown in
Figure 7), revealing that the mildly acidic environment of
the tumor cells could promote the swelling of the micelles
and the release of the loaded drug.
4. Conclusion
In this article, 3-carboxy-5-nitrophenylboronic acid
(CNPBA) shell-crosslinked micelles based on amphiphilic
Dex-b-PLAweredesignedandusedforefficientintracellular
Figure 5. Flow cytometric profiles of MCF-7 cells incubated with
PBS, DOX-loaded PDP-0 (a), and DOX-loaded PDP-3 (b) for 2 h.
Figure 6. Cytotoxicity of PDP-m towards MCF-7 cells and HeLa cells after incubation for 72 h.
Macromol. Biosci. 2014, DOI: 10.1002/mabi.201400251
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Boronic Acid Shell-Crosslinked Dextran-b-PLA Micelles . . .
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Figure 7. Cytotoxicities of DOX-loaded PDP-m and free DOX toward MCF-7 cells and HeLa cells after incubation for 24 h, 48 h, and 72 h.
1
drug deliveries. The copolymers were verified by H NMR
and UV spectra. The CMC, DLS, and TEM data showed that
the shell-crosslinked micelles had pH-responsive proper-
ties. DOX, as a model anticancer drug, was loaded into PDP-
m by a simple dialysis technique. Compared to the
uncrosslinked micelles, the crosslinked micelles showed
lower CMC values and DOX-loaded crosslinked micelles
exhibited a more rapid release rate. The biocompatibilities
of micelles and cellular proliferation inhibition of DOX-
loaded micelles were also investigated with MCF-7 and
HeLa cells. The features identified suggest that these pH-
responsive biodegradable nanocarriers can efficiently load
and deliver DOX into tumor cells and enhance the
inhibition of cellular proliferation in vitro, providing a
favorable platform to construct an efficient drug delivery
system for cancer therapy.
Macromol. Biosci. 2014, DOI: 10.1002/mabi.201400251
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