PH-sensitive polymeric micelles based on amphiphilic polypeptide as smart drug carriers

Article abstract of DOI:10.1002/pola.26830

A series of amphiphilic diblock copolymers having poly(ethylene glycol) (PEG) as one block and a polypeptide as the other block were synthesized by ring-opening polymerization using PEG-amine as a macroinitiator. These polymers were characterized by ¹H-NMR and gel permeation chromatography. The influence of the substitution ratio of tertiary amine-containing groups on the pH sensitivity of the polymers was investigated in detail. Core/shell-structured micelles were fabricated from these polymers using an organic solvent-free method. pH- and concentration-dependent micellization behaviors were investigated by dynamic light scattering and fluorescence microscopy. Micelles loaded with doxorubicin, selected as a model drug, showed restricted drug release at physiological pH but accelerated drug release at tumor extracellular pH. Collectively, our findings suggest that these pH-sensitive micelles might have great potential for cancer therapy applications.

Full text of DOI:10.1002/pola.26830

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pH-Sensitive Polymeric Micelles Based on Amphiphilic Polypeptide as
Smart Drug Carriers
Yi Li, Guang Hui Gao, Doo Sung Lee
Departments of Polymer Science and Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea
Correspondence to: D. S. Lee (E-mail: dslee@skku.edu)
Received 16 April 2013; accepted 13 June 2013; published online 3 July 2013
DOI: 10.1002/pola.26830
ABSTRACT: A series of amphiphilic diblock copolymers having
poly(ethylene glycol) (PEG) as one block and a polypeptide as
the other block were synthesized by ring-opening polymerization
using PEG-amine as a macroinitiator. These polymers were char-
acterized by 1H-NMR and gel permeation chromatography. The
influence of the substitution ratio of tertiary amine-containing
groups on the pH sensitivity of the polymers was investigated in
detail. Core/shell-structured micelles were fabricated from these
polymers using an organic solvent-free method. pH- and
concentration-dependent micellization behaviors were investi-
gated by dynamic light scattering and fluorescence microscopy.
Micelles loaded with doxorubicin, selected as a model drug,
showed restricted drug release at physiological pH but acceler-
ated drug release at tumor extracellular pH. Collectively, our
findings suggest that these pH-sensitive micelles might have
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great potential for cancer therapy applications.
2013 Wiley
Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51,
4175–4182
KEYWORDS: diblock copolymers; drug delivery systems;
micelles
these stimuli, pH is the most widely used owing to the pH
differences between normal tissues and tumor tissues. The
pH values in blood and normal tissues are 7.4; however, the
tumor extracellular pH is as low as 6.5–6.8 and the tumor
endosomal pH could be even lower (5.0–6.5).12–14 The acid-
ity of tumors prompted us to develop intelligent drug deliv-
ery systems based on pH-sensitive polymers. pH-sensitive
polymers that are stable at pH 7.4 but respond to subtle pH
decreases usually have tertiary amine groups or acid-labile
groups on their main chains and/or side chains. Micelles
formed from these polymers are able to retain their payloads
under physiological conditions, but swell or disassociate at
low pH owing to the ionization of amine groups or cleavage
of acid-labile groups.15–18
INTRODUCTION Over the last two decades, polymeric
micelles self-assembled from amphiphilic copolymers have
been extensively investigated and considered as drug car-
riers.1–4 Hydrophobic drugs and/or imaging agents are nor-
mally physically encapsulated or chemically conjugated in
the hydrophobic micelle core, whereas the hydrophilic shell
protects the micelle core and allows the micelles to circulate
in bloodstream. After intravenous administration, these
drug-loaded micelles (usually <200 nm in diameter) can
passively accumulate at solid tumor sites through the well-
known enhanced permeation and retention (EPR) effect.5,6
An efficient drug delivery carrier requires the ability to
retain its payload during circulation in the blood and release
it after reaching tumor sites. However, polymeric micelles
lacking the capacity to undergo a structural change in
response to an appropriate stimulus are incapable of recog-
nizing tumor tissues and releasing drugs in a targeted man-
ner. Thus, it is necessary to develop stimulus-sensitive
polymeric micelles that can efficiently deliver drugs to their
targets.
Previously, several groups have reported pH-sensitive poly-
mers based on poly(b-amino ester) (PAE).19–22 Nanoparticles
formed from these polymers showed fast, sharp demicelliza-
tion behavior upon exposure to an acidic environment and
were evaluated as pH-sensitive carriers of imaging agents or
drugs for detecting and/or treating tumors.23–25 However,
PAE and its derivatives were synthesized by Michael-type
reaction, which made it difficult to control the molecular
weight of the final polymer and resulted in polymers with
broad molecular weight distributions. For biomedical
A number of physical, chemical, and biological stimuli,
including temperature, magnetic fields, pH, redox, light, and
enzymes, have been exploited for the development of intelli-
gent drug delivery systems for cancer therapy.7–11 Among
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applications, polymers with controllable molecular weights
and fine structures are strongly required.
behaviors of these polypeptides were studied by dynamic
light scattering (DLS) and fluorescence spectroscopy under
different pHs and concentration conditions. Finally, the
micelles formed from the pH-sensitive polypeptides were
evaluated as smart carriers for the hydrophobic drug, doxor-
ubicin (DOX).
Stimulus-sensitive synthetic polypeptides, which undergo
reversible secondary conformation transitions and/or hydro-
philic–hydrophobic transitions in response to internal or
external stimuli, are useful biocompatible and biodegradable
polymers with structures that mimic natural proteins.26
These polymers undergo enzymatic degradation and are
mainly synthesized via ring-opening polymerization (ROP) of
a-amino acid N-carboxyanhydrides (NCAs) using initiators
containing primary amines.27,28 The molecular weights of
polypeptides can be controlled by changing the molar ratio
of initiator to monomer. The resulting end primary amine
groups can be used for conjugation of drugs or imaging
agents, or as macroinitiators for further ROPs.
EXPERIMENTAL
Materials
L-Glutamic acid c-benzyl ester, 2-hydroxypyridine, anhydrous
tetrahydrofuran (THF), anhydrous chloroform, and anhy-
drous N,N-dimethylformamide were obtained from Sigma-
Aldrich. Triphosgene and 2-(dibutylamino)ethylamine were
purchased from Tokyo Chemical Industry. Methoxy poly(eth-
ylene glycol) amine (PEG-NH2, Mw 5 5000) was obtained
from SunBio. Hexane, diethyl ether, and ethanol were
obtained from Samchun (Korea). All the chemicals and sol-
vents were used as received.
Bae and coworkers29 have synthesized a pH-sensitive poly-
mer based on poly(L-histidine); however, detailed informa-
tion about pH sensitivity control and pH- or time-dependant
drug release were not reported. In this study, we sought to
synthesize an amphiphilic diblock copolymer with a fine
structure and tunable pH sensitivity for fabricating micelles
as pH-sensitive drug carriers. To accomplish this, we first
synthesized a series of amphiphilic PEG-based polypeptides
using PEG-amine as a macroinitiator. The structures of these
Synthesis of Monomer and Polymers
The synthesis route of the target polymers is shown in
Scheme 1.
Synthesis of c-Benzyl-L-glutamate-N-carboxyanhydride
c-Benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) was
synthesized according to a method reported in the literature
with slight modification.30 Briefly, 10 g of L-glutamic acid
1
polypeptides were confirmed using H-NMR and gel permea-
tion chromatography (GPC). The self-assembling micellization
SCHEME 1 Synthesis route of the PEG-b-PNLG polymers.
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c-benzyl ester and 6.255 g of triphosgene were mixed with
100 mL of anhydrous THF. The reaction was conducted at
50 ꢀC under nitrogen environment. After reaction for 2 h,
the solution was poured into 300 mL of hexane. The crude
product was recrystallized in 1:1 hexane/ethyl acetate (v/v)
for two times. Yield: 73%.
adjusted to 7.4 using 1 mol/L of NaOH aqueous solution,
resulting in the micelle solutions of PEG-b-PNLG copolymers.
Before characterization, all PEG-b-PNLG micelle solutions
were passed through membrane filters (pore size, 0.45 lm;
Millipore). The hydrodynamic radius (size) of PEG-b-PNLG
micelle solutions at different pHs was measured by DLS
(Malvern Instrument, Series 4700) with a helium laser at
633 nm and a digital correlator. The correlation function was
accepted when the difference between the measured and the
calculated baselines was <0.1%. The scattering angle was
fixed at 90ꢀ. The pH- and concentration-dependant micelliza-
tion behavior of PEG-b-PNLG micelles was determined by
fluorescence microscopy at different pHs and polymer con-
centrations using pyrene as a probe. First, a solution of
pyrene in PBS buffer (concentration, 1 3 1026 mol/L) was
prepared. The excitation spectrum of pyrene in the micelle
solutions was recorded at a fixed emission wavelength of
392 nm using a fluorescence spectrometer (AMINCO Bow-
man Series 2).
Synthesis of Poly(ethylene glycol)-block-poly(benzyl-L-
glutamate)
PEG-NH2 and BLG-NCA were dissolved in anhydrous chloro-
form at different molar ratios. The reaction was allowed to
stand for 72 h at room temperature under nitrogen environ-
ment. After the reaction was completed, the solution was
concentrated by a rotary evaporator and precipitated in
excess ethanol/ethyl ether mixture. The obtained polymer
was dried in a vacuum oven for 48 h.
Synthesis of Poly(ethylene glycol)-block-poly[2-
(dibutylamino)ethyl-L-glutamate]
Poly(ethylene glycol)-block-poly(benzyl-L-glutamate) (PEG-b-
PBLG) was dissolved in anhydrous N,N-dimethylformamide
(10 mL/1 g) under nitrogen environment in a round-bottom
flask and immersed in an oil bath at 55 ꢀC. 2-(Dibutylami-
no)ethylamine (103 mol ratio to the ester groups of PEG-b-
PBLG) and 2-hydroxypyridine (53 mol ratio to the ester
groups of PEG-b-PBLG) were subsequently added. Poly(ethyl-
Drug Loading and Releasing
DOX was loaded into PEG-b-PNLG micelles as follows: 1 mg
of DOXꢁHCl and 2 equivalent of triethylene amine were dis-
solved in 2 mL of THF containing 10 mg of PEG-b-PNLG.
After stirring overnight, 10 mL of water was poured into
this solution. After another 3-h stirring, this solution was
dialyzed against large amount of water for 24 h to remove
THF and free DOX. Water was replaced for three times dur-
ing dialysis. The drug-loading efficiency (DLE) and drug-
loading capacity (DLC) were measured by absorbance using
a UV–visible spectroscopy (Lambda Vis 7 spectrophotometer,
Perkin-Elmer, CT, USA) at 479 nm, and were calculated by
ene
glycol)-block-poly[2-(dibutylamino)ethyl-L-glutamate]
(PEG-b-PNLG) copolymers with different substitution ratios
were obtained by varying the reaction time and precipitated
in diethyl ether. The final products were obtained after dry-
ing in vacuum oven for 48 h.
Characterization of Monomer and Polymers
1H-NMR spectra were recorded on Varian Unity Inova
500NB (500 MHz) in deuterated chloroform (CDCl3) contain-
ing 0.03 v/v of tetramethylsilane as a reference at room tem-
perature. The molecular weight (Mn) and polydispersity
index of the polymers were measured by GPC using 1 mg/
mL of LiBr-containing DMF as eluent at 50 ꢀC and a flow
rate of 1.0 mL/min. The molecular weights were calibrated
using PEG standards.
the following equation31
:
DLEð%Þ ¼ ½weight of loaded drug=weight of drug in feedꢂ
3 100%
DLC ðwt %Þ ¼ ½weight of loaded drug=
weight of drug-loaded micelleꢂ 3 100%
For drug-releasing experiments, a solution of DOX-loaded
polymeric micelles was added to cellulose ester membrane
tubes (MW cutoff 5 12,000–14,000 Da) and placed in PBS
buffer solution of different pH values (pH 5 7.4 and 6.4) in
a shaking water bath (80 strokes/min) at 37 ꢀC. Water was
changed periodically. The cumulative release was also meas-
Potentiometric Titration
The titration curves of the PEG-b-PNLG polymers were
obtained by an acid–base titration method. Briefly, 50 mg of
PEG-b-PNLG was added into 50 mL of deionized water at
room temperature and a solution with pH of 3.0. After the
polymer was fully dissolved under vigorous stirring, the
ured by absorbance using
a UV–visible spectroscopy
increase of the solution pH was monitored by a pH meter
(Lambda Vis 7 spectrophotometer, Perkin-Elmer, CT, USA) at
479 nm.
TM
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(Mettler Toledo SevenEasy pH Meter S20) after dropping
a 0.1 mol/L of NaOH aqueous solution (0.05 mL).
RESULTS AND DISCUSSION
Preparation and Characterization of PEG-b-PNLG
Micelles
Synthesis and Characterization of BLG-NCA, PEG-b-PBLG,
and PEG-b-PNLG Polymers
General approaches employed for the synthesis of BLG-NCA,
PEG-b-PBLG, and PEG-b-PNLG are shown in Scheme 1. BLG-
NCA was prepared based on a previously published method,
with some modifications.30 L-Glutamic acid c-benzyl ester
The PEG-b-PNLG micelles were prepared by a simple method
without the use of organic solvents. Polymers were added
into phosphate-buffered saline (PBS) (pH 3.0) at desired con-
centrations. After vigorous stirring for 30 min, clear polymer
solutions were formed. The pH of the polymer solutions was
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block. The degree of polymerization and molecular weight of
PEG-b-PBLG were calculated based on the integrated ratio of
peak f and peak b (characteristic PEG peak).
An aminolysis reaction was employed to synthesize PEG-b-
PNLG by grafting 2-(dibutylamino)ethylamine to the PEG-b-
PBLG backbone (PEG-b-PBLG with a degree of polymerization
of 39 [PEG-b-PBLG39] was employed in consideration of hydro-
phobic/hydrophilic balance) with simultaneous debenzylation.
To prevent chain scission of the polymer backbone (amide
linkages of the polypeptide), we used 2-hydroxypyridine as a
bifunctional catalyst during aminolysis as described previ-
ously.32 The aminolysis conversion at different reaction times
was monitored using 1H-NMR, and the relationship between
the conversion and the reaction time is shown in Figure 2. As
shown in Figure 2, the reaction rate was initially fast, with
40% of benzyl groups replaced by (dibutylamino)ethylamine
after 12 h. However, the reaction rate slowed as the reaction
proceeded, and possibility caused by the decrease of the con-
centration of unreacted benzyl groups at later points in the
reaction. After 72 h, almost all benzyl groups (97%) had been
replaced (Table 2). The complete replacement of benzyl
groups after 72 h was confirmed by 1H-NMR [Fig. 1(c)]. The
characteristic peaks of the benzyl groups (5.1 and 7.3 ppm)
almost disappeared, and peaks h, i, j, and k in Figure 1(c) cor-
respond to the butyl groups in the newly formed PEG-b-PNLG
polymer. The conversion of benzyl groups at different reaction
times is summarized in Table 2. As summarized in Table 2,
the solubility of PEG-b-PNLG polymers was correlated with
the percentage of benzyl groups converted. At conversion per-
centages lower than 63%, PEG-b-PNLG polymers were not
fully soluble in water at any pH. However, at conversion per-
centages higher than 80%, the polymers showed pH-
dependent solubility and could be dissolved in water at low
pH (pH 3.0).
FIGURE 1 1H NMR spectra of BLG-NCA (a), PEG-b-PBLG39 (b),
and PEG-b-PNLG(96%) (c).
and 0.5 equivalents of triphosgene were mixed in anhydrous
THF at 50 ꢀC. Two hours later, a completely homogeneous
solution had formed, indicating the formation of BLG-NCA.
The crude product was obtained by pouring the solution
into excess hexane and was purified by repeated recrystalli-
zation in an ethyl acetate/hexane mixture. The final product
was obtained as a white crystal with high yield, and its
structure and purity were confirmed by 1H-NMR, as shown
in Figure 1(a).
GPC was performed to demonstrate the successful synthesis
of PEG-b-PBLG and PEG-b-PNLG. As shown in Figure 3, both
polymers exhibited unimodal molecular weight distributions
and shorter elution times than the macroinitiator (PEG-amine)
in the GPC traces, indicating the absence of homopolymers
and the formation of diblock copolymers.
PEG-b-PBLG was synthesized by ROP using methoxy PEG
containing an end primary amine as a macroinitiator. The
molecular weight of the PBLG block could be controlled by
varying the molar ratio of monomer to initiator, with a
higher ratio, leading to a longer PBLG block (Table 1). In Fig-
ure 1(b), the peaks at approximately 5.1 ppm (peak f) and
7.3 ppm (peak g) correspond to benzyl groups in PBLG
pH-Buffering Capacities of PEG-b-PNLG Polymers
The pH-buffering properties of PEG-b-PNLG polymers were
assessed using an acid–base titration method. As shown in
Figure 4(a), it is evident that the pH-buffering capacities of
TABLE 1 Feed Ratios and Characteristics of Intermediated Copolymers PEG-b-PBLG
Reaction
Time (h)
Molar Ratio of
Degree of
a
Entries
Initiator/Monomer
Polymerization
Mn
PEG-b-PBLG30
PEG-b-PBLG39
PEG-b-PBLG55
72
72
72
1/30
1/40
1/50
30
39
55
11,570
13,541
17,045
a
Calculated from 1H NMR.
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FIGURE 3 GPC traces of PEG-NH2, PEG-b-PBLG39, and PEG-b-
PNLG(88%).
FIGURE 2 Conversion of benzyl groups in PEG-b-PBLG39 ver-
sus reaction time.
Properties of PEG-b-PNLG Micelles
Polymeric micelles based on PEG-b-PNLG polymers [except
PEG-b-PNLG(40%) and PEG-b-PNLG(63%)] could be simply
fabricated in the absence of organic solvents by increasing
the pH of the polymer solution from 3.0 to 7.4. The pH sen-
sitivities of different PEG-b-PNLG micelles were first investi-
gated by DLS. As shown in Figure 5(a) and summarized in
Table 2, the hydrodynamic sizes of these micelles at pH 7.4
were all small, ranging from 40.0 to 46.0 nm. With decreas-
ing pH, all micelles exhibited an increase in size. This
increase in size is attributable to partial ionization of tertiary
amine groups, which makes the micelle core less hydropho-
bic, causing it to loosen. Further decreasing pH caused the
simultaneous demicellization of the micelles. However, the
starting destabilization pH of PEG-b-PNLG micelles was
related to the conversion of polymer benzyl groups. Micelles
formed from polymers with a lower benzyl group conversion
percentage showed a lower pH threshold for destabilization.
This phenomenon is similar to that observed for pKa values,
and is believed to be caused by the relatively strong hydro-
phobicity of the micelle core formed from PNLG blocks with
a lower conversion of benzyl groups. Figure 5(b) shows the
DLS intensity of PEG-b-PNLG micelles at different pH values.
The scattering intensities of all polymeric micelles decreased
with decreasing pH, and the pH value at which the scattering
PEG-b-PNLG polymers increased with increasing conversion
of benzyl groups. [The pH-buffering capacities of PEG-b-
PNLG(40%) and PEG-b-PNLG(63%) were not evaluated
because they were not soluble in water.] This relationship is
explained by the fact that a higher content of tertiary amine
groups leads to the consumption of more OH2 groups, which
results in stronger pH-buffering capacity. The pKa values of
PEG-b-PNLG polymers were determined as the inflection
points of titration curves. The pKa values of PEG-b-PNLG
polymers (Table 2) also increased with increasing conversion
of benzyl groups. According to the previously published
reports, the pKa values of tertiary amines are related to their
chemical environment, with a more hydrophobic environ-
ment, resulting in lower pKa values.20,33 As benzyl groups
are more hydrophobic than 2-(dibutylamino)ethyl groups,
PEG-b-PNLG polymers with a lower percentage of benzyl
group conversion exhibited lower pKa values. Figure 4(b)
shows the relationship between pKa and reaction time. The
increase in pKa with reaction time showed a similar tend-
ency as the increase in conversion of benzyl groups with
reaction time, such that increases in pKa were almost linearly
related to the conversion percentage [Fig. 4(c)].
TABLE 2 Characteristics and Properties of PEG-b-PNLG Polymers and Micelles
Reaction
Time (h)
Solubility
CMC
Sizeb (nm)
a
Entries
Conversion (%)
Mn
in Water
pKa
(mg/mL)
PEG-b-PNLG(40%)
PEG-b-PNLG(63%)
PEG-b-PNLG(80%)
PEG-b-PNLG(88%)
PEG-b-PNLG(93%)
PEG-b-PNLG(96%)
12
24
36
48
60
72
40
63
80
88
93
96
11,115
11,216
12,823
13,942
12,340
12,011
Insoluble
–
–
–
Not fully soluble
pH dependent
pH dependent
pH dependent
pH dependent
–
–
–
6.32
6.55
6.65
6.69
0.020
0.019
0.019
0.017
46.0
45.2
42.0
40.0
a
Calculated from 1H NMR.
Obtained at pH 7.4 by DLS.
b
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intensity disappeared was same with the pH value, of the
corresponding micelle, at which the size disappeared.
The pH sensitivity of PEG-b-PNLG micelles was further inves-
tigated by fluorescence microscopy using pyrene as a probe.
Pyrene is a relatively hydrophobic dye that preferentially
localizes to a nonpolar environment. The peak emission wave-
length of pyrene dispersed in water is 334 nm, but shifts to
337 nm when pyrene is located in a nonpolar environment,
such as the hydrophobic micelle core. Thus, the micellization/
demicellization behavior of PEG-b-PNLG micelles at different
pH values could be investigated by monitoring the fluores-
cence intensity ratios of pyrene at 334 and 337 nm (I337
/
I334). As shown in Figure 6(a), at pH 7.4, the I337/I334 values
of the PEG-b-PNLG polymers investigated were >1.0, demon-
strating the presence of micelles at this pH. However, with
decreasing pH, the I337/I334 values decreased to <1.0, indicat-
ing the destabilization of micellar structures. Moreover, I337
/
I334 values decreased rapidly below a certain pH value, indi-
cating a sharp micellization/demicellization transition that
implies that these polymeric micelles were quite sensitive to
subtle changes in pH. Figure 6(a) also shows that micelles
formed from polymers with a lower conversion of benzyl
groups had a lower threshold pH for destabilization, which is
FIGURE 4 Titration curves of PEG-b-PNLG polymers (1 mg/mL
in deionized water) (a), relationship of pKa and reaction time (b),
and relationship of pKa and conversion of benzyl groups (c).
FIGURE 6 I337/I334 values of pyrene in different PEG-b-PNLG
polymer solutions (1 mg/mL) at different pHs (a) and fluores-
cence intensity of pyrene at different pHs in 1 mg/mL of PEG-
b-PNLG(88%) polymer solution (b).
FIGURE 5 Size of PEG-b-PNLG polymeric micelles at different
pHs (a) and DLS intensity of these micelles at different pHs (b).
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consistent with the results obtained from DLS results. The fluo-
rescence intensity of pyrene in a 1 mg/mL PEG-b-PNLG(88%)
polymer solution at different pH values is shown in Figure
6(b). At pH values below 6.6, the peak emission wavelength of
pyrene was 334 nm, indicating no micelles formed in this pH
range. In contrast, increasing pH to above 7.0 shifted the peak
emission wavelength of pyrene to 337 nm, indicating the for-
mation of micelles and the localization of pyrene to the micelle
core. Moreover, the fluorescence intensities of pyrene at pH val-
ues above 7.0 were much lower than those at pH values below
6.6, which is owing to the quenching of pyrene fluorescence
owing to the presence of tertiary amine groups in the micelle
core.34
and its fluorescence intensity increased, indicating destabili-
zation of the self-assembled micelles. The relationship
between I337/I334 values of pyrene and polymer concentra-
tions is shown in Figure 7(b); in this figure, the critical
micelle concentration (CMC) is defined as the point at which
the two lines cross. All CMC values of the polymers were
approximately 0.02 mg/mL (Table 2) and were independent
of the conversion of benzyl groups, indicating good stability
of the corresponding polymeric micelles.
DOX Loading and Releasing
As the PEG-b-PNLG(88%) polymer showed suitable pH sensi-
tivity and good stability at pH 7.4, with no significant size
change even after 1 week, micelles formed from this polymer
were chosen as carriers for DOX. DOX was loaded into PEG-
b-PNLG(88%) micelles by pouring excess PBS into a solution
of polymer and DOX in THF, followed by dialysis against PBS
for 24 h at room temperature. DLS measurements showed
that the size of DOX-loaded micelles was 81.8 6 1.6 nm,
which is larger than that of empty micelles. The DLE and
DLC were calculated to be 34.9 and 3.4%, respectively, which
are lower than that of drug-loaded micelles prepared by
thin-film hydration methods.23 We believe that this reduction
in DLE is attributable to the higher solubility of DOX in
THF/PBS than in PBS and drug loss during dialysis. Figure 8
shows the drug-release behavior of DOX-loaded PEG-b-
PNLG(88%) micelles at different pH values (pH 5 7.4 and
6.4). Although the drug-loaded micelles had been dialyzed
before the drug-release experiment, an initial burst of drug
release still occurred at pH 7.4. However, after 2 h, the
release of DOX at pH 7.4 was suppressed, and no more than
30% of drug was released after 24 h. At pH 6.4, the drug-
release rate was much faster than that at pH 7.4 owing to
the destabilization of micelles. After 24 h, about 80% of DOX
had been released, demonstrating the feasibility of using
PEG-b-PNLG(88%) micelles as smart drug carriers that can
rapidly release drug under tumor endosomal pH (5.0–6.5).
The micellization behavior of PEG-b-PNLG micelles was also
investigated at different polymer concentrations. The fluores-
cence intensities of pyrene in PEG-b-PNLG(88%) solutions of
different polymer concentrations are shown in Figure 7(a),
which shows that the peak emission wavelength of pyrene in
high-concentration polymer solutions was 337 nm and fluo-
rescence intensities were low, indicating the presence of
micelles. However, in low-concentration polymer solutions,
the peak emission wavelength of pyrene shifted to 334 nm
FIGURE 7 Fluorescence intensity (a) and I337/I334 values (b) of
pyrene in PEG-b-PNLG(88%) polymer solutions of different
concentrations.
FIGURE 8 Release kinetics of DOX from PEG-b-PNLG(88%)
micelles at pH 7.4 and 6.4 (37 ꢀC).
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12 D. Schmaljohann, Adv. Drug. Deliv. Rev. 2006, 58, 1655–
CONCLUSIONS
1670.
A series of amphiphilic copolymers composed of PEG as a
hydrophilic block and a polypeptide-based polymer as a
hydrophobic block were prepared. The molecular weights of
these polymers could be simply controlled by varying the feed
ratio of macroinitiator to monomer. It was found that the pH
sensitivities of the PEG-b-PNLG polymers could be adjusted by
changing the substitution ratio of tertiary amine-containing
groups, which, in turn, could be controlled by changing the
aminolysis reaction time. DOX-loaded PEG-b-PNLG(88%)
micelles exhibited a pH-responsive drug-release profile, sug-
gesting their potential value in targeting acidic tumors.
13 J. Gu, W.-P. Cheng, J. Liu, S.-Y. Lo, D. Smith, X. Qu,
Z. Yang, Biomacromolecules 2008, 9, 255–262.
14 M. S. Kim, D. S. Lee, Chem. Commun. 2010, 46, 4481–4483.
15 E. S. Lee, K. T. Oh, D. Kim, Y. S. Youn, Y. H. Bae, J. Control.
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16 R. Liu, D. Li, B. He, X. Xu, M. Sheng, Y. Lai, G. Wang, Z. Gu,
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17 W. Chen, F. Meng, F. Li, S.-J. Ji, Z. Zhong, Biomacromole-
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ACKNOWLEDGMENTS
This research was supported by grants from the Basic Science
Research Program through the National Research Foundation
of Korea (NRF) (2010-0027955) and the Bio &#38; Medical
Technology Development Program of the NRF funded by the
Korean government (MEST) (2011-0019391).
20 S. J. Hwang, M. S. Kim, J. K. Han, D. S. Lee, B. S. Kim, E. K.
Choi, H. J. Park, A. S. Kim, Macromol. Res. 2007, 15, 437–442.
21 C. Y. Zhang, Y. Q. Yang, T. X. Huang, B. Zhao, X. D. Guo,
J. F. Wang, L. J. Zhang, Biomaterials 2012, 33, 6273–6283.
22 H. Wang, F. Xu, Y. Wang, X. Liu, Q. Jin, J. Ji, Polym. Chem.
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23 J. Ko, K. Park, Y.-S. Kim, M. S. Kim, J. K. Han, K. Kim, R.-W.
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