Synthesis and physicochemical characterization of amphiphilic triblock copolymer brush containing pH-sensitive linkage for oral drug delivery

Article abstract of DOI:10.1021/la301099q

A novel and well-defined pH-sensitive amphiphilic triblock copolymer brush poly(lactide)-b-poly(methacrylic acid)-b-poly(poly(ethylene glycol) methyl ether monomethacrylate) (PLA-b-PMAA-b-PPEGMA) and its self-assembled micelles were developed for oral administration of hydrophobic drugs. The copolymer and its precursors were synthesized by the combination of activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) and ring-opening polymerization (ROP) techniques. The molecular structures and characteristics were confirmed by GPC, ¹H NMR, and FT-IR. The critical micelle concentration (CMC) values of PLA-b-PMAA-b-PPEGMA in aqueous medium varied from 1.4 to 2.6 mg/L, and the partition equilibrium constant (K_v) of pyrene in micellar solutions ranged from 2.873 × 10⁵ to 3.312 × 10⁵. The average sizes of the self-assembled blank and drug-loaded micelles were 140-250 nm determined by DLS in aqueous solution. The morphology of the micelles was found to be spherical by SEM. Nifedipine (NFD), a poorly water-soluble drug, was selected as the model drug and wrapped into the core of micelles via dialysis method. The in vitro release behavior of NFD from the micelles was pH-dependent. In simulated gastric fluid (SGF, pH 1.2), the cumulative release percent of NFD was relative low, while in simulated intestinal fluid (SIF, pH 7.4), more than 96% was released within 24 h. All the results showed that the pH-sensitive PLA-b-PMAA-b-PPEGMA micelle may be a prospective candidate as oral drug delivery carrier for hydrophobic drugs with controlled release behavior.

Full text of DOI:10.1021/la301099q

Article
pubs.acs.org/Langmuir
Synthesis and Physicochemical Characterization of Amphiphilic
Triblock Copolymer Brush Containing pH-Sensitive Linkage for Oral
Drug Delivery
You Qiang Yang, Wen Jing Lin, Bin Zhao, Xiu Fang Wen, Xin Dong Guo, and Li Juan Zhang*
School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, 510640, P. R. China
ABSTRACT: A novel and well-defined pH-sensitive amphiphilic triblock
copolymer brush poly(lactide)-b-poly(methacrylic acid)-b-poly(poly-
(ethylene glycol) methyl ether monomethacrylate) (PLA-b-PMAA-b-
PPEGMA) and its self-assembled micelles were developed for oral
administration of hydrophobic drugs. The copolymer and its precursors
were synthesized by the combination of activators regenerated by electron
transfer atom transfer radical polymerization (ARGET ATRP) and ring-
opening polymerization (ROP) techniques. The molecular structures and
characteristics were confirmed by GPC, 1H NMR, and FT-IR. The critical
micelle concentration (CMC) values of PLA-b-PMAA-b-PPEGMA in
aqueous medium varied from 1.4 to 2.6 mg/L, and the partition
equilibrium constant (Kv) of pyrene in micellar solutions ranged from
2.873 × 105 to 3.312 × 105. The average sizes of the self-assembled blank
and drug-loaded micelles were 140−250 nm determined by DLS in aqueous solution. The morphology of the micelles was found
to be spherical by SEM. Nifedipine (NFD), a poorly water-soluble drug, was selected as the model drug and wrapped into the
core of micelles via dialysis method. The in vitro release behavior of NFD from the micelles was pH-dependent. In simulated
gastric fluid (SGF, pH 1.2), the cumulative release percent of NFD was relative low, while in simulated intestinal fluid (SIF, pH
7.4), more than 96% was released within 24 h. All the results showed that the pH-sensitive PLA-b-PMAA-b-PPEGMA micelle
may be a prospective candidate as oral drug delivery carrier for hydrophobic drugs with controlled release behavior.
most extraordinary and significant methods.13−16 The different
pH-sensitive polymeric micelles as oral drug carriers can be
designed to be a suitable release for different organs, tissues,
and cells. A successful oral polymeric micellar vehicle should
1. INTRODUCTION
Oral administration is one of the most common routes
preferred by patients owing to its convenience, cost-
effectiveness, flexibility in various chronic treatment regimes,
and high level of patient acceptance.1,2 However, when
delivered orally, many hydrophobic drugs fail to arrive at
their target sites through the gastrointestinal tract, which leads
to low bioavailability, because of a lack of water solubility.3,4
Polymeric core−shell micelles self-assembled from amphiphillic
block copolymers have received much attention as drug
carriers, due to their unique hydropholic inner core, which
can enhance the loading efficiency and provide a desirable
microenvironment for the incorporation of hydrophobic
drugs.5−8 Meanwhile, the outer hydrophilic shell can also
provide a stable interface between the hydrophobic core and
the aqueous environment besides compartmentalization of
hydrophobic drugs. Appropriate micellar size (20−300 nm in
diameter) and a lower critical micelle concentration (CMC) are
important characteristics that can lead to higher biophysical
stability, longer blood circulation time, lower toxic side effects,
and prevention of recognition and uptake by the reticuloendo-
thelial system (RES).9−12
remain stable in the stomach (pH 1.0−3.0), which maintains in
a closed form and prevents the release of drugs from the core of
micelles, while releasing the molecularly dispersed drug in the
intestine (pH 5.1−7.8), thereby preventing burst drug release
and avoiding precipitation in the upper part of gastrointestinal
tract.17−21 Poly(acrylic acid) (PAA) and poly(methacrylic acid)
(PMAA), bearing the carboxylic group with pKa around 5−6,
are the two representative and frequently pH-sensitive
polymers used in oral DDS. They are commonly accepted as
bioadhesive and safe polymers, as the ionic carboxyl of PAA or
PMAA in the micellar corona can facilitate mucoadhesion. This
enhances the residence time of the drug-entrapped micelles in
the gastrointestinal tract and ensures complete drug dissolution,
and eventually maximizes drug absorption in the small
intestine.22,23
The objective of the current work is to design and synthesize
a novel well-defined pH-sensitive amphiphilic triblock copoly-
mer brush PLA-b-PMAA-b-PPEGMA and its self-assembled
An ever-increasing number of types of stimuli (pH,
temperature, redox, light, ultrasound, and enzyme) are being
explored on the environmentally sensitive micelles as smart
drug delivery systems, and the pH-sensitivity is still one of the
Received: March 14, 2012
Revised: May 3, 2012
Published: May 8, 2012
© 2012 American Chemical Society
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glycol (112 mL, 2 mol) and triethylamine (11.13 mL, 80 mmol) were
introduced at 0 °C. Then, 2-bromopropionyl bromide (0.989 mL, 80
mmol) was added dropwise to the cold solution for a period of 2 h.
The reaction was continued at 0 °C for 2 h and then heated to 40 °C
for another 5 h. The reaction mixture was cooled, added to 500 mL of
deionized water, and extracted with chloroform three times, and then
the chloroform layer was washed successively with dilute HCl,
saturated NaHCO3, and water. The organic phase was dried through
MgSO4 overnight followed by rotary evaporation to remove the
solvent. The colorless liquid product was collected by distillation
micelles for oral administration of hydrophobic drugs. The
hydrophobic PLA block has been widely used in biomedical
applications for its excellent biocompatibility and bioabsorb-
ability in vivo.24 It formed the micelle core, maintained high
drug loading content and controlled the drug release rate.
PMAA block was designed to be a block copolymer structure,
which made the core more sensitive to the pH change of the
environment when the polymer self-assembled into micelles.
The hydrophilic PPEGMA with short side chains, known to be
nonimmunogenic, nonantigenic and nontoxic, distributed on
the surface of the self-assembled micelles, providing a compact
steric protective layer to maintain the stability of micelles
during biological circulation.25 Nifedipine, a dihydropyridine
calcium-channel blocker, has received much attention for its
utility in the treatment of hypertension, angina, and myocardial
infarction.26 It is therefore desirable to develop nifedipine
controlled-release chemotherapeutic agents to reduce the side
effects, prevent toxicity, extend half-time, and improve patient
compliance. Herein, nifedipine was used as the model drug and
was encapsulated into PLA-b-PMAA-b-PPEGMA assembled
micelles. The effect of the PMAA block length on the pH
sensitivity of the nifedipine-loaded micelles was studied. Figure
1 shows the schematic micellization and pH-dependent drug
release process of PLA-b-PMAA-b-PPEGMA in aqueous
solution.
1
under reduced pressure and characterized by H NMR (CDCl3, as
solvent): −C(CH3)2Br δ = 2.05 ppm (6H), CH2−CH2−OH δ = 3.85
ppm (2H), CH2−CH2−OH δ = 4.29 ppm (2H), CH2−CH2−OH δ =
2.33 ppm (1H).
2.3. ARGET ATRP of tBMA Initiated by HEBriB. Poly(tert-butyl
methacrylate) (HO-PtBMA-Br) was synthesized by ARGET
ATRP.28,29 Typically, a previously dried 50 mL Schlenk flask equipped
with a magnetic stirring bar was charged with CuBr2 (11.2 mg, 0.05
mmol), and the flask was evacuated and flushed with argon three
times. Anhydrous toluene (18 mL) and tBMA (5.715 mL, 35.2 mmol)
were added to the flask using degassed syringes, and the mixture was
homogenized by ultrasonic dispersion 10 min. Then, PMDETA (0.210
mL, 1 mmol) was introduced into the flask via syringe, and stirred 10
min until the Cu/PMDETA catalyst complex was formed. Sn(Oct)2
(0.162 mL, 0.5 mmol) in toluene (2 mL) was then added to reduce
the deactivator. Finally, HEBriB (0.165 mL, 1 mmol) was added and
the flask was placed in a preheated oil bath maintained at 90 °C for 2
h. The flask was removed from the oil bath and cooled to room
temperature. After the solvent was removed on a rotary evaporator, the
reaction mixture was diluted with THF and passed through a neutral
alumina column to remove the catalyst. Finally, HO-PtBMA-Br was
precipitated by adding the solution into 10-fold excess of water/
methanol (1:1, v/v) mixture, filtered, and dried under vacuum for 24
h.
2.4. ROP of D,L-LA in the Presence of HO-PtBMA-Br.
Poly(lactide)-b-poly(tert-butylmethacrylate) (PLA-b-PtBMA-Br) di-
block copolymer was synthesized by ROP.30,31 The freshly recrystal-
lized momomer D,L-LA (2.5 g, 0.5 mmol) and macroinitiator HO-
PtBMA-Br (Mn = 4549, 0.5 mmol) were placed in a flame-dried 50 mL
Schlenk flask with a magnetic stirring bar, and the Schlenk flask was
evacuated and backfilled with argon three times. Subsequently, a
required amount of Sn(Oct)2 (0.081 mL, 0.5 mmol) in toluene (20
mL) was added, and three freeze−pump−thaw cycles were performed
to remove any oxygen from the solution. The flask was filled with
argon and placed into a thermostatted oil bath at 100 °C for 24 h.
Afterward, the flask was removed from the oil bath and cooled to a
room temperature followed by rotary evaporation to remove the
solvent. The crude polymer was dissolved in approximately 30 mL
THF and added dropwise to 300 mL water/methanol (1:1, v/v)
mixture to precipitate the product, which was collected and dried
under vacuum for 24 h.
2.5. Synthesis of PLA-b-PtBMA-b-PPEGMA. The synthetic
procedure of poly(lactide)-b-poly(tert-butyl methacrylate)-b-poly-
(poly(ethylene glycol) methyl ether monomethacrylate) (PLA-b-
PtBMA-b-PPEGMA) is similar to that of HO-PtBMA-Br. Briefly, a
previously dried 50 mL Schlenk flask equipped with a magnetic stirring
bar was charged with PLA-b-PtBMA-Br (Mn = 6516, 0.6 mmol) and
CuBr2 (6.7 mg, 0.03 mmol). After three degassed and backfilled cycles
with argon, anhydrous toluene (18 mL), macromonomer PEGMA
(0.343 mL, 12 mmol), and PMDETA (0.063 mL, 0.3 mmol) were
injected into the reaction flask with syringe. After stirring for 15 min, a
solution of Sn(Oct)2 (0.097 mL, 0.3 mmol) in toluene (2 mL) was
added and three freeze−pump−thaw cycles were conducted. The flask
was transferred into a thermostatted oil bath at 70 °C for 24 h. After
the solvent was removed on a rotary evaporator, the reaction mixture
was diluted with THF, passed through a neutral alumina column, and
precipitated with n-hexane. The final dried powder products were
obtained after vacuum drying for 24 h.
Figure 1. Scheme of drug loading and pH-dependent release from
PLA-b-MAA-b-PPEGMA micelles.
2. EXPERIMENTAL SECTION
2.1. Materials. tert-Butyl methacrylate (tBMA, TCI-EP) were
washed with sodium hydroxide solution (10%), distilled from calcium
hydride, and stored under argon at −20 °C. D,L-Lactide (D,L-LA,
Aldrich) was recrystallized from ethyl acetate three times and dried at
room temperature under reduced pressure. PEGMA (Mn = 300 Da,
Aldrich, 99%) was purified by passing through a column filled with
neutral alumina to remove inhibitor. Dichloromethane (DCM, 99.8%,
Sigma-Aldrich), triethylamine (TEA, 99%, Sigma-Aldrich), and
toluene were dried over CaH2 and distilled under nitrogen flow.
Nifedipine (NFD, 99%, Sigma-Aldrich), pyrene (99%, Aldrich), ethyl
2-bromoisobutyrate (EBriB, 99%, Aldrich), N,N,N,N′,N′-pentamethyl
diethyl enetriamine (PMDETA, 99%, Aldrich), tetrahydrofuran
(THF), trifluoroacetic acid (TFA), n-hexane, CuBr2, methanol,
acetone, and all other reagents were used as received.
2.2. Synthesis of Difuctional Initiator 2-Hydroxylethyl-2-
Bromoisobutyrate (HEBriB). HEBriB was synthesized as follows:27
To a flame-dried 150 mL Schlenk flask with a magnetic stirring bar,
which was evacuated and flushed with argon three times, ethylene
2.6. Hydrolysis of PLA-b-PtBMA-b-PPEGMA. Poly(lactide)-b-
poly(methacrylic acid)-b-poly(poly(ethylene glycol) methyl ether
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Scheme 1. Synthetic Route of PLA-b-PMAA-b-PPEGMA Copolymer
monomethacrylate) (PLA-b-PMAA-b-PPEGMA) was prepared by a
selective hydrolysis of PLA-b-PtBMA-b-PPEGMA to remove the tert-
butyl groups from PtBMA unit. In a typical experiment, 1500 mg of
PLA-b-PtBMA-b-PPEGMA (Mn = 7276, 0.12 mmol) was dissolved in
15 mL of DCM. After cooling to 0 °C with ice/water bath, 2.64 mL of
TFA (5-fold molar of tert-butyl groups) was slowly added with
vigorous stirring. The mixture was stirred at 0 °C for 30 min and then
carried out at room temperature for 4 h. After evaporating and
removing all the solvents by rotary evaporation, the residues were
dissolved in 5 mL of THF and precipitated into 50 mL of n-hexane
(10-fold of THF) three times. The resulting brush-like copolymer
PLA-b-PMAA-b-PPEGMA was collected by filtration and dried in a
vacuum oven at 40 °C for 48 h.
2.7. Determination of Critical Micelle Concentrations (CMC).
The formation of micelles self-assembled from PLA-b-PMAA-b-
PPEGMA brush in an aqueous phase was confirmed by the
fluorescence technique with a pyrene probe. The fluorescence of
pyrene is well-known to be sensitive to change in the microenviron-
ment. When the environment of pyrene changes from polar to
nonpolar, the excitation spectra of pyrene are altered, and the peak
shifts from 335 to 337 nm in the excitation spectra of pyrene as the
copolymer concentration increases. In brief, the pyrene solution in
acetone was added to deionized water (pH 6.5) until a pyrene
concentration of 12 × 10−5 M was reached. Acetone was subsequently
removed by rotary evaporator at 30 °C for 24 h. The final
concentration of pyrene was adjusted to 6 × 10−7 M in the copolymer
solution. The concentrations of polymer solution were varied from
dissolved in 10 mL of ethanol under vigorous vortexing. The
concentration of NFD at 237 nm was recorded with reference to a
calibration curve of pure NFD/ethanol solution. The LC and EE of
NFD were calculated using the following formulas 1 and 2,
respectively. All samples were analysized in triplicate.
weight of NFD‐loaded drug
weight of total polymer and drug in feed
LC(%) =
EE(%) =
× 100%
(1)
weight of NFD‐loaded drug
× 100%
weight of NFD in feed
(2)
2.9. In Vitro Release of NFD from Micelles. The in vitro NFD
release properties from the PLA-b-PMAA-b-PPEGMA assembled
micelles were determined as follows: 5 mg of NFD-entrapped micelle
was suspended in 3 mL of simulated gastric fluid (SGF, 0.15 M HCl,
0.05 M KCl, pH 1.2) or simulated intestinal fluid (SIF, 8 g NaCl, 0.2 g
KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, 1 L water, pH 7.4) and then
placed in a pre-swollen cellulose membrane bag (MWCO 3500−
4000). The whole bag then was immersed into 30 mL of SGF or SIF
buffer and shaken in a 37 °C water bath (Dissolution Tester RCZ-8B,
TDTF, China) at 100 rpm. At specified time intervals, a 3 mL (Ve)
sample was taken out and replaced by 3 mL fresh buffer to maintain
the total volume. The concentrations of NFD in different samples
were determined using UV−vis spectrophotometer. The cumulative
drug release percent (Er) was calculated based on the eq 3. The in vitro
experiments were repeated three times, and all samples were analyzed
in triplicate to get the final release curves.
0.0001 to 0.1 mg/mL. At last, the solutions were kept at room
temperature in the dark for 24 h to reach the solubilization equilibrium
between pyrene and copolymer.
V ∑n−1 Ci + V Cn
e
0
1
Er(%) =
× 100
2.8. Preparation of Blank and NFD-Loaded Micelles. The
blank or NFD-loaded PLA-b-PMAA-b-PPEGMA micelles were
prepared according to the diafiltration method. In a typical experiment,
NFD (0−10 mg) and PLA-b-PMAA-b-PPEGMA (40 mg) were
dissolved in 40 mL of DMF and stirred for 4 h at room temperature,
followed by dialysis against deionized water (pH 6.5) for 24 h at 20 °C
using a dialysis bag (molecular weight cutoff, MWCO 3500−4000).
The deionized water was changed every 2 h for the first 6 h and then
replaced every 6 h. After dialysis, half of the blank micelle solution was
used to study the pH-responsive behavior of the micelle solution by
the addition of NaOH or HCl (0.01 M) solution, and the remaining
blank and NFD-loaded micelles were filtered by a membrane filter
(0.45 μm pore) to remove aggregated particles and collected by
freeze−drying to obtain dried product. The received white powder was
stored at −20 °C until further experiments.
mNFD
(3)
where mNFD represents the amount of NFD in the micelle (mg), V0 is
the whole volume of the release media (V0 = 33 mL), and Cn
represents the concentration of NFD in the nth sample (mg).
2.10. Characterization. The number average molecular weight
(Mn) and polydispersity index (Mw/Mn) were determined by gel
permeation chromatography (GPC) adopting an Agilent 1200 series
GPC system equipped with a LC quant pump, PL gel 5 mm 500 Å, 10
000 Å, and 100 000 Å columns in series, and RI detector. The column
system was calibrated with a set of monodisperse polystyrene
standards using HPLC-grade THF as mobile phase with a flow rate
of 1.0 mL/min at 30 °C.
1H NMR spectra measurements were executed on a NMR
spectrometer (Bruker AVANCE III 400, Switzerland) operating at
250 MHz, using deuterated chloroform (CDCl3) or deuterated
dimethyl sulfoxide (DMSO-d6) as solvent and tetramethyl silane
(TMS) as an internal standard. The temperature was 25 °C.
NFD loading content (LC) and entrapment efficiency (EE) were
determined by UV−vis spectrophotometer (UV-2450, Shimadzu,
Japan). One milligram of the NFD-loaded micelle powder was
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Table 1. GPC and H NMR Data of PLA-b-PMAA-b-PPEGMA Brushes and Its Precursors
Article
1
a
b
c
c
d
d
entry
sample
HO-PtBMA-Br1
[M]0/[I]0
Con(%)
DP
Mn,NMR
Mn,GPC
Mw/Mn
1
2
3
4
5
6
7
8
35:1
40:1
20:1
--
80.5
39.4
13.4
100
28.1
15.8
2.7
4216
6488
7291
5708
4643
7349
8218
6566
4549
6516
7276
5585
4910
7160
8364
6549
1.46
1.48
1.42
1.36
1.51
1.46
1.28
1.32
PLA-b-PtBMA-Br1
PLA-b-PtBMA-b-PPEGMA1
PLA-b-PMAA-b-PPEGMA brush1
HO-PtBMA-Br2
28.1
31.2
18.8
2.9
45:1
40:1
20:1
--
69.3
43.0
14.5
100
PLA-b-PtBMA-Br2
PLA-b-PtBMA-b-PPEGMA2
PLA-b-PMAA-b-PPEGMA brush2
32.3
a
b
c
Molar ratio of the initial monomer and initiator concentration. Determined by 1H NMR. Calculated by 1H NMR end group analysis.
Determined by GPC.
d
FT-IR spectra of PLA-b-PMAA-b-PPEGMA and its precursors were
which was carried out in toluene at 100 °C for 24 h. In the
second-staged ARGET ATRP reaction, PLA-b-PtBMA-b-
PPEGMA was prepared by using PLA-b-PtBMA-Br with
bromoester terminal groups as macroinitiator. The reaction
was carried out in toluene at 70 °C for 24 h in the presence of
the same catalyst/ligand/reducing agent with HO-PtBMA-Br.
The amphiphilic triblock PLA-b-PtBMA-b-PPEGMA was
selectively hydrolyzed employing TFA as acid agent in DCM
to acquire the target PLA-b-PMAA-b-PPEGMA brush, whose
favorite reaction time of hydrolysis was 4 h.32,33 With the
hydrolysis underway, the numbers of tert-butyl groups were
gradually reduced and the solution became turbid (slight
yellow), in respect that PLA-b-PMAA-b-PPEGMA brush is
insoluble in DCM.
The molecular weights of PLA-b-PMAA-b-PPEGMA brush
and its precursors were determined by GPC and summarized in
Table 1. The molar ratio of the initial monomer and initiator
concentration from brush1 and brush2 were 35:1 and 45:1,
respectively, resulting in different lengths of PMAA block,
which affects the in vitro release behavior of the nifedipine-
loaded micelles. Figure 2 (entries 1−4, Table 1) shows the
obtained in a transmission mode on a FT-IR spectrophotometer
(Nicolet Nexus for Euro, America) under ambient condition. Samples
were ground with KBr and then compressed into pellets. The spectra
were taken from 400 to 4000 cm−1. Typically, 32 scans at a resolution
of 8 cm−1 were accumulated to obtain one spectrum.
Fluorescence spectra were obtained using a fluorescence spec-
trophotometer (F-4500, Hitachi, Japan) and the excitation spectra of
polymer/pyrene solutions were scanned from 300 to 350 nm at room
temperature, with an emission wavelength of 373 nm and bandwidth
of 0.2 nm.
The micelle size (Dh) and distribution (PDI) were determined by
dynamic light scattering (DLS, Malven Zetasizer Nano S, U.K.). The
dialysis solutions were filtered through 0.45 μm pore size filters and
the measurements were conducted in a 1.0 mL quartz cuvette, using a
diode laser of 800 nm at 25 °C, and the scattering angle was fixed at
90°. The zeta potentials were determined by electrophoretic
measurement carried out with the same Malven Zetasizer Nano S
instrument and the measurements were performed three times for
each sample at 25 °C. The Dh and zeta potential were received from
the average of three measurement results.
Morphologies of blank and drug-loaded micelles were investigated
by scanning electron microscopy (SEM, LEO 1530 VP, Germany).
The sample was fixed on an aluminum stub as a thin film and coated
with gold before observation. SEM was operated at an accelerating
voltage of 5 kV and a magnification of 10 000× or 20 000× in the
transmission electron mode.
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization of the PLA-b-
PMAA-b-PPEGMA Brush. PLA-b-PMAA-b-PPEGMA brush
was synthesized via a combination of ARGET ATRP and ROP
followed by selective hydrolysis of the tert-butyl groups of
tBMA, as illustrated in Scheme 1. First, HEBriB was
synthesized in the excess of ethylene glycol. Then, HEBriB
was utilized as difunctional initiator for the ARGET ATRP of
tBMA and subsequently ROP of D,L-LA to get PLA-b-PtBMA-
Br. PLA-b-PtBMA-Br was then used to initiate the ARGET
ATRP of PEGMA to form PLA-b-PtBMA-b-PPEGMA. Finally,
PLA-b-PMAA-b-PPEGMA was harvested after the selective
cleavage of the tert-butyl groups.
Figure 2. GPC traces of PLA-b-PMAA-b-PPEGMA brush1 and its
In the first-staged ARGET ATRP reaction, Cu/PMDETA
complex was utilized as catalyst system. A sufficiently large
excess of reducing agent Sn(Oct)2 not only reduces CuII to CuI
but is also responsible for scavenging oxygen and radical
inhibitors. The monomer tBMA was polymerized in toluene at
90 °C with a conversion over 65% in 2 h and well-defined
polymer with a narrow distribution on the condition that the
molar ratio of Sn(Oct)2/CuBr2 was 10/1. By using hydroxy-
terminated HO-PtBMA-Br as macroinitiator and Sn(Oct)2 as
catalyst, PLA-b-PtBMA-Br was synthesized through ROP of
D,L-LA. The molar ratio HO-PtBMA-Br and D,L-LA was 40/1,
precursors.
GPC traces of the serial polymer products using the same HO-
PtBMA-Br1 macroinitiator. It can be seen that the resultant
copolymers are shifted toward higher weight with increasing
molecular weight of successive blocks (entries 1−3, Table 1 and
Figure 2), while the molecular weight of the polymers decreases
slightly after hydrolysis (entry 4, Table 1 and Figure 2). The
molecular weights determined by GPC conformed to the
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theoretical values, and the Mw/Mn values were lower than 1.55
for the controlled/living polymerization.34−36
Mn(PLA−b−PMAA−b−PPEGMA)
= DP × 86 + DP × 144 + DP
× 300
The chemical structures and formations of the products were
PMAA
PLA
PPEGMA
1
1
further determined by H NMR and FT-IR. The H NMR
spectra of PLA-b-PMAA-b-PPEGMA brush1 (solvent, DMSO-
d6) and its percursors (solvent, CDCl3) were depicted in Figure
3. In Figure 3A, the signals at 0.9−1.1, 1.42, and 1.7−1.9 ppm
(11)
+ 211
1
The evidence of H NMR spectra reported in Table 1 is in
good agreement with the GPC values, indicating good control
of the polymerization and preparation of well-defined
copolymers.
As exhibit in the FT-IR spectra, the absorbance of -OH, C
H, CO, and [-(CH3)3-] with stretching and bending bands in
HO-PtBMA-Br (Figure 4A) occurs at 3446 cm−1, 2979 cm−1,
Figure 3. 1H NMR spectra of HO-PtBMA-Br1 (A), PLA-b-PtBMA-Br1
(B), PLA-b-PtBMA-b-PPEGMA1 (C), and PLA-b-PMAA-b-PPEGMA
brush1 (D).
Figure 4. FT-IR spectra of HO-PtBMA-Br1 (A), PLA-b-PtBMA-Br1
(B), PLA-b-PtBMA-b-PPEGMA1 (C), and PLA-b-PMAA-b-PPEGMA
brush1 (D).
are ascribed, respectively, to -CH3, [-(CH3)3-], and -CH2- in
the tBMA unit, whereas the signals of -CH- and -CH3- protons
of PLA block can be found in peaks at 5.17 and 1.58 ppm
(Figure 3B). As observed in Figure 3C, the characteristic
PEGMA peaks at 3.28, 3.65, and 4.30 ppm due to -OCH3,
-OCH2−CH2O-, and -COO−CH2- protons, respectively. After
hydrolysis, the tert-butyl group (1.42 ppm) disappears while the
carboxyl group (12.30 ppm) emerges, which can be seen in
Figure 3D.
1723 cm−1, 1368 cm−1, and 848 cm−1, respectively.
Furthermore, when the ring D,L-LA opens, stretching vibration
are clearly observed at 3508 cm−1 and 1760 cm−1, which are
attributed to the PLA -OH and CO stretching bands (Figure
4B). As Figure 4C shows, the characteristic PEGMA stretching
vibration of C−H and C−O- are observed at 2888 cm−1 and
1137 cm−1. For Figure 4A and C, the band ascribed to the C
C are not detected at 1640 cm−1, supporting the formation of
HO-PtBMA-Br and PLA-b-PtBMA-b-PPEGMA.39,40 A broad
absorption band with a characteristic range of 3300−3500 cm−1
is the stretching vibration of −OH in a carboxylic acid group,
which reflects the hydrolysis of the tBMA (Figure 4D).
3.2. CMC Value of PLA-b-PMAA-b-PPEGMA Brush.
CMC value of PLA-b-PMAA-b-PPEGMA and the hydro-
phobicity of the hydrophobic microdomain of the self-
assembled micelles were determined by the fluorescence
probe technique using pyrene as a fluorescence probe.41,42
The CMC value was determined from the threshold
concentration, where the intensity ratio I337/I335 begins to
increase markedly. The CMCs of PLA-b-PMAA-b-PPEGMA
brush1 and PLA-b-PMAA-b-PPEGMA brush2 given in Figure 5
were determined to be 2.6 and 1.4 mg/L, respectively, which
decreased with an increasing amount of hydrophobic PLA
block, and results were summarized in Table 2. The results
showed that a strong tendency for the copolymers bearing
longer hydrophobic block to be prone to formation of micelles
in aqueous solution. The CMCs of two copolymers were much
lower than those of common surfactant, suggesting that the
The degree of polymerization (DP) of PtBMA, PLA, and
1
PPEGMA can be calculated from the H NMR end group
analysis according to the following formulas:37,38
DP
= Ia/Ib × 2/3
(4)
(5)
(6)
(7)
PtBMA
DP = Id/Ia × 9 × DP
PLA
PtBMA
DP
= Ie/Id × 1/10 × DP
PPEGMA
PLA
DP
= Ih/Id × DP
PMAA
PLA
where Ia, Ib, Id, Ie, and Ih are the intensities of the peaks at 1.42,
2.05, 5.17, 3.65, and 12.3 ppm, respectively.
The number-average molecular weights (Mn) can be thus
obtained by the following equations:
Mn(HO−PtBMA−Br) = DP
× 142 + 211
PtBMA
(8)
Mn(PLA−b−PtBMA−Br) = DP × 144 + Mn(HO−PtBMA−Br)
PLA
(9)
Mn(PLA−b−PtBMA−b−PPEGMA)
= DP × 300 + Mn(PLA−b−PtBMA−Br)
(10)
PPEGMA
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Figure 5. Plot of I337/I335 ratios as a function of logarithm of PLA-b-
PMAA-b-PPEGMA concentrations.
Figure 6. Plot of (F − Fmin)/(Fmax − F) vs concentration of PLA-b-
PMAA-b-PPEGMA brushes in aqueous solution.
Table 2. Composition, CMC, and Kv of the Copolymers
PPEGMA assembled micelles were prepared by diafiltration
method against deionized water (pH 6.5). The size and size
distribution (PDI) of the obtained blank and NFD-loaded
micelles were measured by DLS, and the results are shown in
Table 3. The size of blank micelle was about 140 nm and
increased slightly to be about 150 nm after being loaded with
NFD for PLA-b-PMAA-b-PPEGMA brush1 (entries 1−4, Table
3). For PLA-b-PMAA-b-PPEGMA brush2, the size increased
from 214 to 250 nm after being entrapped with NFD (entries
5−8, Table 3). The NFD-loaded micelles showed a larger size
than the blank micelles, which suggested that NFD was
incorporated into the micelles effectively. The micelles
maintained a narrow unimodal distribution, with a PDI
between 0.200 and 0.400, suggesting a good physical
performance of the assembled micelles.
Zeta-potential measurement was used to further investigate
the properties of the micelles. The blank and NFD-loaded
micelles possess negative charges (−15 to −25 mV) due to
COO− groups in the PMAA chains and the potential is
proportional to the amount of PMAA in the micelles (Table 3).
These results suggested that the PLA-b-PMAA-b-PPEGMA
brush micelles could be mucoadhesive, which facilitates the
bioadhesion between the micelle carriers and intestinal
epithelial cells.48
CMC
Kv
a
a
a
sample
xPLA
xPPEGMA
xPMAA
(mg/L) (×105)
PLA-b-PMAA-b-
PPEGMA brush1
0.398
0.141
0.424
2.6
1.4
2.873
3.312
PLA-b-PMAA-b-
PPEGMA brush2
0.412
0.132
0.425
a
1
Determined by H NMR.
micelles formed from PLA-b-PMAA-b-PPEGMA brush were
relatively stable in solution, even after extreme dilution by the
larger volume of systemic circulation in the body.43
The hydrophobicity of the inner core of self-aggregated
micelles is one important factor in its application as a drug
carrier. In this study, the partition equilibrium constant (Kv) of
pyrene in the hydrophobic core of PLA-b-PMAA-b-PPEGMA
brush micelles was calculated according to the method reported
by Wilhelm et al.44 and Kim et al.45,46 This method calculates
Kv based on the assumption of a simple equilibrium distribution
between the micellar phase and the water phase. The method
can be simplified as eq 12
(F − Fmin)/(Fmax − F) = KvxC/1000ρ
(12)
where Fmin and Fmax correspond to the average magnitude of
the intensity ratio I337/I335 in the flat region of low and high
polymer concentration ranges, respectively, and F is the I337
/
The variations of the size and zeta potential of the blank
micelles as a function of pH were studied, as shown in Table 4.
At higher pH above 6.3, the sizes of the micelles were increased
because the PMAA segments protonated gradually, resulting in
the increasing of hydrophilicity of PMAA and the swelling of
the micelle. As can be also seen in Table 4, the zeta potential of
the micelles presented higher negative charges when pH was
above 6.3. When the pH values were between 6.3 and 4.5, the
sizes were relative small due to the compact micelle structure
assigned to PMAA segments deprotonated. However, the sizes
increased sharply with pH decreasing below 2; this may be due
to the fact that the micelles start to aggregate because of strong
deionization of the micelles and intermolecular hydrogen bond
formation.49
The morphology of the micelles was further examined using
SEM. Figure 7 showed the SEM images of NFD-loaded PLA-b-
PMAA-b-PPEGMA micelles, which demonstrated that the
micelles were spherical in shape. It is of importance to note that
the average sizes shown in SEM images were nearly consistent
with the DLS results.
I335 in the intermediate concentration range in Figure 6. C is the
concentration of PLA-b-PMAA-b-PPEGMA brush. x is the
weight fraction of hydrophobic PLA in the brush, which is
0.398 and 0.412 for PLA-b-PMAA-b-PPEGMA brush1 and
PLA-b-PMAA-b-PPEGMA brush2, respectively, shown in Table
2. ρ is the density of the PLA core of micelles, which is assumed
to take the same value as bulk PLA (=1.25).47 The Kv values for
pyrene were obtained from the slope of the graph by plotting a
graph of (F − Fmin)/(Fmax − F) versus the concentration of
PLA-b-PMAA-b-PPEGMA brush as shown in Figure 6. Thus,
the Kv values of brush1 and brush2 were determined to be 2.873
× 105 and 3.312 × 105 as summarized in Table 2. The effect of
a longer hydrophobic PLA block is evident in the higher Kv, as a
result, indicating the stronger hydrophobicity of the micellar
cores. This is related to the hydrophilic/hydrophobic balance in
the triblock copolymer, which consequently affects the micelle
packing ability.
3.3. Characterization of the Blank and NFD-Loaded
Micelles. All the blank and NFD-loaded PLA-b-PMAA-b-
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Table 3. Size, Size Distribution, Zeta Potential, LC, and EE of NFD-Loaded Micelles
entry
polymer (mg)
NFD (mg)
feed ratio (%)
size (nm)
PDI
zeta potential (mV)
LC (%)
EE (%)
a
1
2
3
4
5
6
7
8
40
0
0
140
151
141
155
214
233
257
237
0.389
0.417
0.436
0.431
0.238
0.232
0.280
0.348
−22.0
−24.6
−19.5
−23.7
−17.4
−19.1
−19.0
−19.2
--
--
a
40
6
15
20
25
0
4.61
5.48
5.62
--
32.25
28.99
23.84
--
a
40
8
a
40
10
0
b
40
b
40
6
15
20
25
5.03
6.07
6.84
35.27
32.30
29.36
b
40
8
b
40
10
a
b
PLA-b-MAA-b-PPEGMA brush1. PLA-b-MAA-b-PPEGMA brush2.
Table 4. Size and Zeta Potential of PLA-b-PMAA-b-
PPEGMA Micelles As a Function of pH
between the drug and the micelle core. In this case, the
solubilization capacity may increase to a saturation level with
increasing drugs. As a result, the EE in the micelles will
decrease.50,51
PLA-b-MAA-b-PPEGMA
PLA-b-MAA-b-PPEGMA
brush2
brush1
3.4. In Vitro Release of NFD from PLA-b-PMAA-b-
PPEGMA Brush Micelles. The release behavior of NFD at the
fixed amount of drug (entries 4 and 8, Table 3) from the
micelles with different lengths of PMAA block were evaluated
at 37 °C in SGF (pH 1.2) and SIF (pH 7.4), as shown in Figure
8. For both brushes, the release rate was relatively low at acidic
zeta
potential
(mV)
zeta
potential
(mV)
size
size
pH
(nm)
PDI
pH
(nm)
PDI
8.73
7.44
6.30
5.42
4.46
3.53
2.42
1.46
194
188
136
132
129
208
343
611
0.389
0.416
0.402
0.220
0.361
0.413
0.148
--
−25.1
−24.6
−21.6
−20.2
−18.3
−13.2
−2.8
8.62
7.42
6.35
5.56
4.60
3.48
2.33
1.41
238
231
209
203
184
216
385
708
0.283
0.233
0.261
0.312
0.129
0.226
0.325
--
−27.6
−22.5
−17.1
−16.4
−13.0
−10.1
−3.6
--
--
The LC and EE values of NFD-loaded micelles were
illustrated in Table 3. It can be seen that weight ratios of drug
to polymer had a profound influence on the LC and EE,13 the
LC increased with the increase of drug fed for both PLA-b-
PMAA-b-PPEGMA brush micelles. Also, as for the same drug/
polymer ratio, the copolymer micelles with longer-length PLA
block and higher Kv exhibited enhanced drug entrapping ability.
The larger the core of micelle assembled from more
hydrophobic PLA content, the larger the amounts of drug
that could be encapsulated into copolymer, which resulted in an
increased LC. As for EE, an increase in the feed amount of
NFD decreased its EE. It may be due to a strong interaction
Figure 8. In vitro drug release profiles of NFD-loaded PLA-b-PMAA-b-
PPEGMA brush assembled micelles.
Figure 7. SEM images of NFD-loaded PLA-b-PMAA-b-PPEGMA brush1 (A) and brush2 (B) assembled micelles.
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pH (pH 1.2), with about 66−73% of NFD released after 24 h.
On the other hand, the amount of NFD released accelerated
significantly in the presence of pH 7.4; more than 96% of NFD
was released within 24 h, due to the swelling of micelle
structure caused by the stronger protonation of carboxyl groups
of PMAA at higher pH conditions. The release rate of NFD was
increased by improving the PMAA block ratio in the PLA-b-
PMAA-b-PPEGMA. The cumulative release of PLA-b-PMAA-b-
PPEGMA brush2 micelle is higher (about 5%) than that of
PLA-b-PMAA-b-PPEGMA brush1 micelle in 12 and 24 h. Such
a drug release pattern from the PLA-b-PMAA-b-PPEGMA
micelles will ensure progressive and complete drug dissolution
in the intestinal tract as well as maximize its release in the small
intestine to offer the largest area for drug absorption, which can
reduce the drug leakage in the stomach. The overall in vitro
drug release results demonstrate that these pH-responsive
micelles self-assembled from PLA-b-PMAA-b-PPEGMA show
promise as delivery vehicles for the efficient administration of
various hydrophobic drugs via the oral route. Encouraged by
these promising results, further studies exploring better
performance of pH-sensitive polymeric micelles by adjusting
their molecular architecture are underway in our laboratory.
Research Funds for the Central Universities (No.
2011ZM0041).
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AUTHOR INFORMATION
■
Corresponding Author
*Tel/Fax: +86-20-87112046, E-mail: celjzh@scut.edu.cn.
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ACKNOWLEDGMENTS
■
This work was financially supported by National Natural
Science Foundation of China (No. 21176090), Team Project of
Natural Science Foundation of Guangdong Province (No.
S2011030001366), Guangzhou Municipal Bureau of Science
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