Supramolecular copolymer micelles based on the complementary multiple hydrogen bonds of nucleobases for drug delivery

Article abstract of DOI:10.1021/bm200155t

Novel supramolecular copolymer micelles with stimuli-responsive abilities were successfully prepared through the complementary multiple hydrogen bonds of nucleobases and then applied for rapid intracellular release of drugs. First, both adenine-terminated poly(-caprolactone) (PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U) were synthesized. The supramolecular amphiphilic block copolymers (PCL-A:U-PEG) were formed based on multiple hydrogen bonding interactions between PCL-A and PEG-U. The micelles self-assembled from PCL-A:U-PEG were sufficiently stable in water but prone to fast aggregation in acidic condition due to the dynamic and sensitive nature of noncovalent interactions. The low cytotoxicity of supramolecular copolymer micelles was confirmed by MTT assay against NIH/3T3 normal cells. As a hydrophobic anticancer model drug, doxorubicin (DOX) was encapsulated into these supramolecular copolymer micelles. In vitro release studies demonstrated that the release of DOX from micelles was significantly faster at mildly acid pH of 5.0 compared to physiological pH. MTT assay against HeLa cancer cells showed DOX-loaded micelles had high anticancer efficacy. Hence, these supramolecular copolymer micelles based on the complementary multiple hydrogen bonds of nucleobases are very promising candidates for rapid controlled release of drugs.

Full text of DOI:10.1021/bm200155t

ARTICLE
pubs.acs.org/Biomac
Supramolecular Copolymer Micelles Based on the Complementary
Multiple Hydrogen Bonds of Nucleobases for Drug Delivery
†
‡
†
,†
‡
‡
‡
‡
Dali Wang, Yue Su, Chengyu Jin, Bangshang Zhu,* Yan Pang, Lijuan Zhu, Jinyao Liu, Chunlai Tu,
‡
,†,‡
Deyue Yan, and Xinyuan Zhu*
†
Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China
‡
School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University,
8
00 Dongchuan Road, Shanghai 200240, People’s Republic of China
S Supporting Information
b
ABSTRACT: Novel supramolecular copolymer micelles with
stimuli-responsive abilities were successfully prepared through
the complementary multiple hydrogen bonds of nucleobases
and then applied for rapid intracellular release of drugs. First,
both adenine-terminated poly(ε-caprolactone) (PCL-A) and
uracil-terminated poly(ethylene glycol) (PEG-U) were synthe-
sized. The supramolecular amphiphilic block copolymers
(
PCL-A:U-PEG) were formed based on multiple hydrogen bonding interactions between PCL-A and PEG-U. The micelles self-
assembled from PCL-A:U-PEG were sufficiently stable in water but prone to fast aggregation in acidic condition due to the dynamic
and sensitive nature of noncovalent interactions. The low cytotoxicity of supramolecular copolymer micelles was confirmed by
MTT assay against NIH/3T3 normal cells. As a hydrophobic anticancer model drug, doxorubicin (DOX) was encapsulated into
these supramolecular copolymer micelles. In vitro release studies demonstrated that the release of DOX from micelles was
significantly faster at mildly acid pH of 5.0 compared to physiological pH. MTT assay against HeLa cancer cells showed DOX-loaded
micelles had high anticancer efficacy. Hence, these supramolecular copolymer micelles based on the complementary multiple
hydrogen bonds of nucleobases are very promising candidates for rapid controlled release of drugs.
’
INTRODUCTION
pH at tumor sites has been considered as an ideal stimulus for the
selective release of anticancer drugs in tumors to achieve tumor-
targeted drug delivery. Thus, micelles based on acid-labile
During the past decades, core/shell polymeric micelles self-
22
assembled from amphiphilic copolymers in water have emerged
1
ꢀ6
covalent bonds such as acetal, hydrazone, cis-acotinyl, and
as one of the most promising drug delivery systems. The inner
hydrophobic core of the micelles serves as a microenvironment
for incorporating hydrophobic drugs by physical entrapment,
while the outer hydrophilic shell maintains a hydration barrier to
provide a stable interface between the hydrophobic core and the
external medium. Block copolymer micelles for drug delivery
application are usually associated with several advantages. First,
they improve the solubility and bioavailability of hydrophobic
2
3ꢀ26
ortho-ester are designed and applied in drug delivery field.
27
For example, Zhong et al. prepared acid-sensitive micelles
based on the block copolymer of poly(ethylene glycol) (PEG)
and polycarbonate with acetal moieties, in which the hydrolysis
of acetals under acidic condition led to the release of loaded
anticancer drugs. Kataoka et al. reported pH-sensitive poly-
meric micelles by attaching DOX to a PEGylated polyaspartate
block copolymer via an acid-labile hydrazone bond. In addition,
Heller and co-workers have exploited ortho-ester as acid-labile
linkages for design of pH-responsive micelles for drug delivery.
These pH-responsive micelles based on acid-sensitive covalent
bonds exhibit sustained release of drugs over tens of hours via a
7
,8
28
9
,10
drugs in water.
Furthermore, the nano size and narrow size
29
distribution of micelles can effectively avoid renal clearance and
11
nonspecific reticuloendothelial uptake. Therefore, these mi-
celles significantly protect the incorporated drugs from fast
degradation and elimination in the body and prolong drug
3
0,31
1
2,13
diffusion-controlled mechanism under acidic conditions.
circulation time after intravenous administration.
However, for cancer therapy, it is often more desirable to
accomplish rapid drug release after micelles arriving at the
pathological sites, which may enhance the therapeutic efficacy
Recently, stimuli-responsive micelles have gained widespread
attention for programmable delivery systems in which the release
of drugs can be readily controlled by exerting an appropriate
3
2
1
4ꢀ19
as well as reduce probability of drug resistance in cells.
stimulus (e.g., pH, temperature, glutathione, etc).
Among
all these stimuli, acidic pH as an internal stimulus is particularly
fascinating because tumor sites and inflammatory tissues as well
as the intracellular compartments such as endosomes and lyso-
Received: February 1, 2011
Revised:
March 2, 2011
20,21
somes of cells have a more acidic environment.
The acidic
Published: March 03, 2011
r 2011 American Chemical Society
1370
dx.doi.org/10.1021/bm200155t
|Biomacromolecules 2011, 12, 1370–1379

Biomacromolecules
ARTICLE
Therefore, it is necessary to design and exploit novel polymeric
micelles with rapid response to acidic environment.
at 27 °C for 24 h under an argon atmosphere. The polymerization was
terminated by adding excess acetic acid. DMSO was evaporated under
reduced pressure, and the residue was precipitated into cold diethyl
Compared to conventional covalent-linked polymers, supra-
molecular polymers based on noncovalent interactions have been
found to be more sensitive to external stimuli, which offers a new
route for design of drug delivery systems with rapid response
ether twice. The product was dried under vacuum to a constant weight.
1
Yield: 86%. H NMR (400 MHz, CDCl , 298 K) δ ppm: 8.3 (1H,-
3
NdCHN-), 7.8 (1H, dNCHdN-), 5.6 (2H, -NH ), 4.4 (2H,-
2
3
3,34
NCH
COOCH
per repeating unit, -COdCH CH -), 1.5ꢀ1.8 (polycaprolactone, 2H
2
CH
2
-), 3.9ꢀ4.2 (polycaprolactone, 2H per repeating unit,-
abilities.
Among various noncovalent interactions, hydrogen
2
-), 3.7 (2H, -NCH CH
2
2
-), 2.1ꢀ2.4 (polycaprolactone, 2H
bonding is very sensitive to pH variation. Therefore, it can be
imaged that if the hydrophobic and hydrophilic blocks are linked
through strong multiple hydrogen bonding interactions, a novel
supramolecular copolymer with highly pH-sensitive abilities can
be obtained. Ascribed to the perfect combination of amphiphilic
and pH-responsive properties, self-assembled micelles with rapid
controlled release property may become realizable.
2
2
per repeating unit, -COCH
CH CH
-), 1.2ꢀ1.4 (polycaprolactone, 2H per repeating unit,-
COCH CH CH -).
2 2
CH -, 2H per repeating unit, -COO-
2
2
2
2
2
Preparation of PEG-U. PEG-U was prepared by coupling uracil to
PEG. In a typical procedure: NaOH (0.27 g, 6.72 mmol) and PEG (12.0
g, 2.4 mmol) in anhydrous CH
2
Cl
2
(100 mL) were cooled in an
Cl solution
In biological systems, multiple hydrogen bonding interactions
occur in the adenineꢀuracil (A-U), adenineꢀthymine (A-T),
iceꢀwater bath with stirring. After stirring for 30 min, a CH
2
2
35
(40 mL) of p-toluenesulfonyl chloride (TsCl; 0.82 g, 4.3 mmol) was
and guanineꢀcytosine (G-C) base pairs in DNA and RNA. In
the present work, the complementary adenineꢀuracil (A:U)
base pair between hydrophobic PCL-A and hydrophilic PEG-U
was employed to construct supramolecular amphiphilic block
copolymers (PCL-A:U-PEG). Benefiting from their amphiphili-
city and noncovalent connection, PCL-A:U-PEG could self-
assemble into micelles with pH-responsive abilities in aqueous
solution. These novel stimuli-responsive micelles not only
exhibited similar properties to conventional micelles from cova-
lent-linked copolymers, but also responded rapidly to acidic
stimulus. Moreover, the cytotoxicity and cellular uptake of the
drug-loaded micelles were also evaluated using MTT assay, flow
cytometry, and confocal laser scanning microscopy (CLSM).
slowly added to the reaction mixture and stirred for an additional 24 h at
0
°C. The resulting mixture was poured into water and extracted with
CH Cl . The combined organic extracts were washed with water and
saturated sodium chloride (NaCl), dried over anhydrous magnesium
sulfate (MgSO ), filtered, and the filtrate was evaporated. PEG-sulfanilic
2
2
4
acid ester was obtained with a yield of 89%. After that, PEG-sulfanilic
acid ester (4.0 g, 0.8 mmol), uracil (0.18 g, 1.6 mmol), potassium
carbonate (K CO ; 0.22 g, 1.6 mmol) were dissolved in 60 mL of DMF
2
3
in a round-bottomed flask and reacted at 80 °C for 8 h. The resultant
mixture was poured into water and extracted with CH Cl . After drying
2
2
over MgSO , most of the solvent was removed by evaporation. The
4
residue was precipitated into cold diethyl ether and dried under vacuum
1
to a constant weight. Yield: 85%. H NMR (400 MHz, CDCl , 298 K) δ
3
ppm: 8.4 (1H, -CONHCO-), 7.4 (1H, -NCHCH-), 5.6 (1H, -CHCH-
CO-), 3.9 (2H, -CH CH N-), 3.5ꢀ3.7 (PEG, 2H per repeating unit,-
2
2
’
EXPERIMENTAL SECTION
OCH
2
CH
2
-, 2H per repeating unit, -OꢀCH -), 3.4 (3H, CH
CH N-),
8.0 (CH O-), 67.8 (-CH CH N-), 69.7 (-CH CH O-), 71.2 (CH OCH -),
2
CH
2
3
-
1
3
OCH
2
-). C NMR (100 MHz, CDCl
3
, 298 K) δ: 47.04 (-CH
2
2
Materials. N,N-Dimethylformamide (DMF), dimethylsulfoxide
5
1
(DMSO), and methylene dichloride (CH
2
Cl
2
) were dried over calcium
3
2
2
2
2
3
2
00.2 (-CHCHCO-), 146.3 (-CHCHCO-), 150.9 (-NCONH-), 163.7
hydride for 48 h and then distilled before use. Toluene was dried by
refluxing with the fresh sodium-benzophenone complex under nitrogen
gas and distilled just before use. ε-Caprolactone (ε-CL, 99%, J&K) was
dried over calcium hydride for 24 h and then purified by distillation
under reduced pressure prior to use. Uracil (U, 99%, Sigma), adenine (A,
(
-CHCONH-).
Characterization. Both H NMR and C NMR spectra were
recorded on Bruker AVANCEIII 400 spectrometer with CDCl
DMSO-d , and 1,1,2,2-tetrachloroethane-d as solvents. The molecular
1
13
3
,
6
2
9
9%, J&K), triethylaluminum (AlEt
ethylene carbonate (99%, Fluka) were all used as received. Mono-
methoxy poly(ethylene glycol) (PEG) with M = 5000 g/mol was
3
, 0.6 M in heptane, J&K), and
weights and polydispersity index (PDI) were determined by gel
permeation chromatography/multiangle laser light scattering (GPC-
MALLS). The gel permeation chromatography system consisted of a
Waters degasser, a Waters 515 HPLC pump, a 717 automatic sample
injector, a Wyatt Optilab DSP differential refractometer detector, and a
Wyatt miniDAWN multiangle laser light scattering detector. Three
chromatographic columns (styragel HR3, HR4, and HR5) were used in
series. Tetrahydrofuran (THF) was used as the mobile phase at a flow
rate of 1 mL/min at 30 °C. The refractive index increment dn/dc was
determined with Wyatt Optilab DSP differential refractometer at
690 nm. Data analysis was performed with Astra software (Wyatt
Technology). Fourier transform infrared (FTIR) spectra were recorded
on a Paragon 1000 instrument by KBr sample holder method. Absor-
bance measurements were carried out using Perkin-Elmer Lambda 20/
2.0 UV/vis spectrometer. The calibration curve of absorbance against
different concentrations of DOX was made at 485 nm. Dynamic light
scattering (DLS) measurements were performed with a Malvern Zeta-
sizer Nano S apparatus equipped with a 4.0 mW laser operating at λ =
633 nm. All samples were measured at a scattering angle of 173°. The
data were the mean of three tests. Transmission electron microscopy
(TEM) studies were performed with a JEOL JEM-100CX-II instrument
at a voltage of 200 kV. Samples were prepared by drop-casting micelle
solutions onto carbon-coated copper grids and then air-drying at room
temperature before measurement.
n
purchased from Fluka and dried by azeotropic distillation in the presence
of dry toluene. Doxorubicin hydrochloride (DOX HCl) was purchased
from Beijing Huafeng United Technology Corporation and used as
received. Clear polystyrene tissue culture treated 12-well and 96-well
plates were obtained from Corning Costar. All other reagents and
solvents were of analytical grade and used as received unless mentioned.
3
0
Synthesis of 9-(2 -Hydroxyethyl) Adenine (HEA). A mixture
containing adenine (2.11 g, 15.6 mmol), ethylene carbonate (1.40 g,
15.7 mmol), and a trace of sodium hydroxide (NaOH) in DMF
(
100 mL) was heated at 120 °C for 2 h. After filtration, the DMF was
removed under reduced pressure and the residue was recrystallized from
1
ethanol to give out some white powder. Yield: 70%. H NMR (400 MHz,
DMSO-d , 298 K) δ ppm: 8.14 (s, 1H, ArH), 8.08 (s, 1H, ArH), 7.23 (s,
2
6
2 2 2
H, NH ), 5.03 (s, 1H, OH), 4.19 (t, 2H, CH ), 3.74 (q, 2H, CH ).
Synthesis of PCL-A. Under an argon atmosphere, HEA (0.1260 g,
ꢀ4
7
.0 ꢁ 10 mol), DMSO (30 mL), and a toluene solution of AlEt
3
(0.1
mol/L, 0.5 mL) were added to a fresh flamed and argon-purged round-
bottomed flask. The solution was stirred at 27 °C for 0.5 h and then
toluene was evaporated under reduced pressure. After repeating this
procedure three times, the mixture was cooled to 0 °C and 5 mL of
caprolactone (0.046 mol) was added. The resulting mixture was stirred
1
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Biomacromolecules
ARTICLE
Formation of PCL-A:U-PEG Micelles and Their Critical
Micelle Concentration. Typically, both PEG-U (10 mg, 0.002 mol)
and PCL-A (11 mg, 0.002 mol) were dissolved in THF (2 mL) at room
temperature. After that, the polymer solution was added dropwise into
well in 1 mL complete DMEM and cultured for 24 h. Then the DOX-
loaded micelles dissolved in DMEM culture medium at a final DOX
concentration of 9 μg/mL were added to different wells and the cells
were incubated at 37 °C for 5, 30, 60, and 180 min. Thereafter, samples
were prepared for flow cytometry analysis by removing the cell growth4
media, rinsing with PBS, and treating with trypsin. Data for 1.0 ꢁ 10
gated events were collected and analysis was performed by means of a
BD FACSCalibur flow cytometer and CELLQuest software.
10 mL of deionized water under stirring with a magnetic bar. Then the
THF was removed by vacuum distillation with a rotary evaporator. The
micelle solution was dialyzed against deionized water for 24 h (MWCO =
2000), during which the water was renewed every 4 h.
The critical micelle concentration (CMC) was determined using 1,6-
For the CLSM studies, HeLa cells were seeded in six-well plates at 1 ꢁ
5
diphenyl-1,3,5-hexatriene (DPH) as UV probe by monitoring the
absorbance at 313 nm. The concentration of block copolymer was
10 cells per well in 1 mL of complete DMEM and cultured for 24 h,
followed by removing culture medium and adding DOX-loaded micelles
(1 mL of DMEM medium) at a final DOX concentration of 9 μg/mL.
The cells were incubated at 37 °C for predetermined intervals. Subse-
quently, the cells were washed with PBS and fixed with 4% paraformal-
dehyde for 30 min at room temperature, and the slides were rinsed with
PBS for three times. Finally, the slides were mounted and observed with
a LSM510 META.
ꢀ
4
varied from 1.0 ꢁ 10 to 0.2 mg/mL and the DPH concentration was
ꢀ
6
fixed at 5.0 ꢁ 10 mol/L. The absorbance spectra of all solutions were
recorded using Perkin-Elmer Lambda 20/2.0 UV/vis spectrometer.
Preparation of DOX-Loaded Micelles. Briefly, DOX HCl and
3
an equal molar amount of triethylamine (TEA) were dissolved in DMSO
and added to a THF solution of PEG-U with an equivalent molar PCL-A
at a theoretical drug loading content of 10 wt %. Then the mixture was
added slowly to 5 mL of phosphate-buffered saline (PBS, 50 mM, pH
Activity Analyses. The cytotoxicity of DOX-loaded micelles and
free DOX against HeLa cells was evaluated in vitro by MTT assay. HeLa
3
7
.4). After being stirred for an additional 4 h, the solution was dialyzed
cells were seeded into 96-well plates with a density of 8 ꢁ 10 cells per
against deionized water for 24 h (MWCO = 2000), during which the
water was renewed every 4 h. For determination of drug loading content,
the DOX-loaded micelle solution was lyophilized and then dissolved in
DMF. The UV absorbance at 485 nm was measured to determine the
DOX concentration.
well in 200 μL of medium. After 24 h of incubation, the culture medium
was removed and replaced with 200 μL of a medium containing serial
dilutions of DOX-loaded micelles. The cells were grown for another 48
h. Then, 20 μL of 5 mg/mL MTT assays stock solution in PBS was
added to each well. After incubating the cells for 4 h, the medium
containing unreacted dye was removed carefully. The obtained blue
formazan crystals were dissolved in 200 μL per well DMSO and the
absorbance was measured in a BioTek Elx800 at a wavelength of 490 nm.
Drug loading content (DLC) and drug loading efficiency (DLE) were
calculated according to the following formula:
DLCðwt%Þ ¼ ðweight of loaded drug=weight of polymerÞ ꢁ 100%
’
RESULTS AND DISCUSSION
DLEð%Þ ¼ ðweight of loaded drug=weight of drug in feedÞ
Synthesis and Characterization of PCL-A and PEG-U. End-
ꢁ
100%
functionalized polymers can be synthesized via direct polymer-
ization using a functional initiator or through the postmodifica-
In Vitro Release Study. A total of 6 mL of DOX-loaded micelles
was transferred to a dialysis bag (MWCO = 2000). It was immersed in
36ꢀ40
tion of polymers with reactive terminal moieties.
Scheme 1
7
5 mL of phosphate buffer (pH 7.4) or acetate buffer (pH 5.0) solutions
gives the synthesis route of nucleobase-teminated PCL and PEG.
First, HEA was formed through the reaction of adenine with
ethylene carbonate according to previous reports, which was
confirmed by NMR measurements. Then adenine-terminated
PCL was synthesized via the ring-opening polymerization
in a shaking water bath at 37 °C to acquire sink conditions. At
predetermined time intervals, 2 mL of the external buffer was withdrawn
and replenished with an equal volume of fresh media. The amount of
released DOX was analyzed with fluorescence (QM/TM/IM Steady-
State and Time-Resolved Fluorescence Spectrofluorometer) measure-
ment (excitation at 480 nm). The release experiments were conducted in
triplicate. The results were the average data.
41
(
ROP) of ε-CL using HEA as the initiator and AlEt as the
3
1
catalyst. The obtained PCL-A was well characterized by H NMR
and GPC-MALLS. Typical H NMR spectra of PCL-A is shown
1
Cell Culture. NIH/3T3 normal cells (a mouse embryonic fibroblast
cell line) and HeLa cancer cells (a human uterine cervix carcinoma cell
line) were cultured in DMEM (Dulbecco’s modified Eagle’s medium)
supplied with 10% FBS (fetal bovine serum), and antibiotics (50
units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a
in Figure 1A. Figure 2 gives the GPC curves of PCL-A with
different molecular weights and the characterization data are
summarized in Table 1. In addition, number-average molecular
1
weights are also calculated from the H NMR integration ratios
of the methylene signal on the PCL polymer backbone
2
humidified atmosphere containing 5% CO .
(
-CH OCO-, 3.9ꢀ4.2 ppm) to the signal of methylene con-
2
Cytotoxicity Measurements of Micelles. The relative cyto-
toxicity of PCL-A:U-PEG micelles against NIH/3T3 cells was estimated
by MTT viability assay. In the MTT assay, NIH/3T3 cells were seeded
nected with adenine (-N-CH CH -, 4.4 ppm). These results are
in agreement with those reported in previous literature.
2 2
4
1
4
PEG-U was obtained by the reaction of PEG-sulfanilic acid
ester with excess uracil in the presence of K CO . The chemical
into 96-well plates with a density of 1 ꢁ 10 cells per well in 200 μL of
2
3
medium. After 24 h of incubation, the culture medium was removed and
replaced with 200 μL of a medium containing serial dilutions of micelles.
The cells were grown for another 48 h. Then, 20 μL of 5 mg/mL MTT
assays stock solution in PBS was added to each well. After incubating the
cells for 4 h, the medium containing unreacted dye was removed
carefully. The obtained blue formazan crystals were dissolved in 200
μL per well DMSO and the absorbance was measured in a BioTek
Elx800 at a wavelength of 490 nm.
1
13
structure of PEG-U is confirmed by the H NMR and C NMR
spectra shown in Figures 1B and S1. The peaks at 3.9 and 8.4
ppm are ascribed to the protons of CH (-CH CH N-) con-
2
2
2
nected with uracil and NH (-CONHCO-) on the uracil, respec-
13
tively, which demonstrates the generation of PEG-U. The C NMR
results further demonstrate the successful grafting of uracil on PEG.
Hydrogen Bonding Interactions between PCL-A and PEG-U.
The formation of complementary multiple hydrogen bonds be-
tween PCL-A and PEG-U was analyzed by variable-temperature
Cellular Uptake of Micelles by HeLa Cells. The cellular uptake
experiments were performed on flow cytometry and CLSM. For flow
cytometry, HeLa cells were seeded in six-well plates at 6 ꢁ 10 cells per
5
1
H NMR spectroscopy for the blend of two polymers with an
1
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Biomacromolecules
ARTICLE
Scheme 1. Synthetic Route to Adenine-Terminated Poly(ε-caprolactone) (PCL-A) and Uracil-Terminated Poly(ethylene glycol)
PEG-U)
(
1
Figure 1. H NMR spectra of adenine-terminated poly(ε-caprolactone) (PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U): (A) PCL-A,
B) PEG-U (400 MHz, CDCl ).
(
3
equivalent mole in 1,1,2,2-tetrachloroethane-d . The temperature
resonance moves upfield systematically from 8.7 to 7.9 ppm when
the temperature is raised from 25 to 100 °C. The gradual decrease
in the NH resonance with the increase of temperature can be
attributed to the dissociation of the complementary hydrogen
2
dependence of the NH and NH proton chemical shift of the PCL-
2
A and PEG-U complex (1:1) at 2.5 wt % in 1,1,2,2-tetrachlor-
oethane-d is shown in Figure 3. The chemical shift of the NH
2
1
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Biomacromolecules
ARTICLE
Figure 2. Gel permeation chromatography (GPC) curves of adenine-
terminated poly(ε-caprolactone) (PCL-A): (A) PCL25-A, (B) PCL40-A,
(C) PCL49-A.
1
Figure 3. H NMR NH and NH
2
chemical shifts of the adenine-
terminated poly(ε-caprolactone) (PCL-A) and uracil-terminated poly-
(
ethylene glycol) (PEG-U) complex (1:1) as a function of temperature.
Table 1. Characterization of Adenine-Terminated Poly-
Sample was allowed to equilibrate for 10 min at each temperature (400
MHz, 1,1,2,2-tetrachloroethane-d2).
(
ε-caprolactone) (PCL-A) Polymers
a
b
b
b
polymers
PCL-A
M
n
M
n
M
w
PDI
1
( H NMR)
(GPC)
(GPC)
(GPC)
Table 2. Properties of the PCL-A:U-PEG Micelles
1
2
3
2800
3600
4500
2870
4590
5630
2890
4950
6470
1.01
1.08
1.15
a
b
b
sample
CMC (mg/mL)
diameter (nm)
PDI
ꢀ
2
2
PCL25-A:U-PEG114
PCL40-A:U-PEG114
PCL49-A:U-PEG114
6.5 ꢁ 10
3.9 ꢁ 10
1.8 ꢁ 10
142
156
172
0.097
0.093
0.139
a
1
b
ꢀ
Determined by H NMR. Determined by gel permeation chroma-
tography/multiangle laser light scattering (GPC-MALLS) using THF as
the eluent.
ꢀ2
a
Critical micelle concentration (CMC) was determined by UV/vis
spectrometer. Diameter and polydispersity index (PDI) of PCL-A:U-
b
PEG micelles were determined by dynamic light scattering (DLS).
41,42
bonds.
However, as soon as the sample is cooled from 100 to
2
5 °C, the NH resonance returns to its original position at 8.7 ppm.
Similarly, the NH resonance exhibits the same change in the range
2
U-PEG block copolymers were prepared by dialysis method and
the resulting micelles had a yield of 92% (weight of resulting
micelles/weight of copolymers in feed). The CMC of these
supramolecular copolymers were investigated by UV/vis spectra
using DPH as a hydrophobic probe. The relationship of the
content of PCL with the CMC is listed in Table 2. As the content
of PCL in copolymers increases, the CMC gradually decreases.
To further study the properties of supramolecular copolymer
micelles, both DLS and TEM measurements were performed.
DLS results show that all of the PCL-A:U-PEG micelles exhibit
unimodal size distribution with the mean diameter from 142 to
172 nm and the detailed data are summarized in Table 2.
Apparently, the average diameter of the micelles increases as
the PCL increases from M = 2.8 k to M = 5.6 k with the same
of 5.74ꢀ5.35 ppm. All of these data suggest that complementary
hydrogen bonds generate between PCL-A and PEG-U, and the
supramolecular diblock copolymers are thermoreversible in 1,1,2,2-
tetrachloroethane-d2.
To further evaluate the hydrogen bonding interactions be-
tween PCL-A and PEG-U, FTIR was performed at various
temperatures (from 25 to 160 °C). Figure S2 shows the partial
IR spectra of PCL-A and PEG-U complexes in the bulk state. The
ꢀ
1
bands between 1750 and 1710 cm come from the CdO
stretching vibration. It can be found that the CdO stretching
ꢀ
1
peak shifts to higher frequency from 1724 to 1734 cm as the
temperature raises from 25 to 160 °C. When the sample is cooled
from 160 to 25 °C, the CdO stretching peak returns to its
n
n
ꢀ
1
original position at 1724 cm . According to previous literature,
PEG block. This indicates that a long PCL block can enhance its
assembly and lead to a large core. The morphologies of supra-
molecular copolymer micelles for PCL -A:U-PEG visualized
the CdO stretching bands shift to lower frequencies upon
43
hydrogen bond formation. It is concluded that the comple-
mentary hydrogen bonds dissociate with increasing temperature.
The results of the FTIR spectra suggest that polymer chains are
linked through complementary hydrogen bonds to form a
supramolecular diblock copolymer with thermoreversibility.
Formation of Micelles and pH-Triggered Destabilization.
PCL-A and PEG-U could form supramolecular diblock copolymer
throughcomplementary hydrogen bondinginteractions. Owingto
the presence of the hydrophobic PCL-A and hydrophilic PEG-U
domains, the supramolecular amphiphilic block copolymer self-
assembled into micelles in aqueous solution. Micelles of PCL-A:
49
114
by TEM are shown in Figure 4B1. The copolymer aggregates into
approximate spherical micelles in aqueous solution and the
micelle sizes determined by TEM are in accordance with the
data from DLS in Figure 4A1.
The hydrogen bonding interactions between hydrophobic
PCL-A core and hydrophilic PEG-U shell made copolymer
micelles unstable at acidic pH. To evaluate the responsive ability,
the copolymer micelles were treated with pH 5.0 acetate buffer
(50 mM) and the particle sizes were followed by DLS measure-
ments at different time intervals. Figure 5A shows that PCL -A:
4
9
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Figure 4. Characterization of PCL49-A:U-PEG114 and doxorubicin (DOX)-loaded micelles. (A) The size distribution of PCL49-A:U-PEG114 micelles
determined by DLS: (A1) PCL49-A:U-PEG114 micelles and (A2) DOX-loaded copolymer micelles. (B) TEM images of self-assembled micelles: (B1)
PCL49-A:U-PEG114 micelles and (B2) DOX-loaded copolymer micelles.
Figure 5. Characterization of PCL49-A:U-PEG114 micelles: (A) Size change of pH-sensitive micelles over time at pH 5.0 at 25 °C monitored by DLS;
(B) Photographs of copolymer micelles over 48 h (B1) pH = 7.4, (B2) pH = 5.0; (C) The size of PCL49-A:U-PEG114 micelles at different temperatures
determined by DLS. Error bars represent the standard deviation (n = 3).
U-PEG114 micelles aggregate rapidly at pH 5.0. The size of
copolymer micelles increases from 172 nm to about 722 nm in
Thermal Stability of PCL-A:U-PEG Micelles. The stability of
supramolecular block copolymers was studied at different tem-
peratures. As shown in Figure 5C, the effect of temperature on
PCL-A:U-PEG micelles is very slight at the temperature below
60 °C and the average diameter is around 172 nm. When self-
assembled micelles are heated from 60 to 90 °C, the micelle size
reaches 185 nm. It is related to the dissociation of complemen-
tary hydrogen bonds at high temperature. Therefore, the stability
of prepared micelles at physiological temperature offers the
possibility for the introduction to the body with the potential
of therapeutics delivery.
4
h, and reaches over 1000 nm after 24 h. The photographs in
Figure 5B show that the micelle solution becomes turbid at low
pH value after 48 h, while still keeps clear under physiological pH
condition (pH 7.4). The driving force for the change of micelle
size at low pH value is attributed to the protonation of the
nucleobase nitrogen atoms, which leads to the shedding of
hydrophilic PEG shell from the micelles and the aggregation of
44
hydrophobic PCL core. In contrast, no change in micelle size is
observed after 48 h at pH = 7.4.
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Figure 6. Cell viability of NIH/3T3 cells against nanomicelles after
cultured for 48 h with different micelle concentrations.
Figure 8. Flow cytometry histogram profiles of HeLa cells that were
incubated with self-assembled PCL49-A:U-PEG114 micelles containing
core-encapsulated doxorubicin (DOX).
selected as a drug carrier. Hydrophobic DOX was well-encapsu-
lated into the hydrophobic inner cores of the polymeric micelles
(
PCL -A:U-PEG114) with a loading efficiency of about 15%.
49
The theoretical DLC was set 10% and the final DLC of PCL -A:
4
9
U-PEG114 was 1.6%. It should be noted that DOX-loaded
micelles show a lightly increased average size of about 198 nm,
measured by DLS and TEM (Figure 4A2 and B2), wherein the
polydispersity of polymeric micelles is fairly low, indicating
narrow size distribution.
The in vitro release behavior of DOX-loaded micelles was
investigated by dialysis in two different buffered solutions (pH
7
PEG
.4 and 5.0) at 37 °C to assess the feasibility of using PCL -A:U-
4
9
Figure 7. In vitro release profiles of doxorubicin (DOX) from PCL -A:
U-PEG114 micelles at different pH values (7.4 and 5.0) at 37 °C.
4
9
micelles as an anticancer drug delivery carrier. As
14
1
illustrated in Figure 7, the release profiles show that DOX is
released quickly from the micelles in the first stage and then the
drug release could be sustained over a prolonged time. It is
obvious that the DOX release from micelles at pH 5.0 is much
faster than the release at pH 7.4, indicating a pH-dependent drug
release profile. At pH 7.4, the release rate of DOX is low with less
than 40% of the DOX released in 40 h, while a noticeably
increased release rate of DOX is observed at pH 5.0 with more
than 65% of the DOX released within 5 h. This is in accordance
with the previous observation that PCL -A:U-PEG micelles
In Vitro Cytotoxicity of PCL-A:U-PEG Micelles. The cyto-
toxicity of PCL-A:U-PEG micelles was evaluated by MTT assay
using NIH/3T3 normal cells. The MTT assay is based on the
ability of a mitochondrial dehydrogenation enzyme in viable cells
to cleave the tetrazolium rings of the pale-yellow MTT and form
formazan crystals with dark-blue color. Figure 6 shows the cell
viability after 48 h incubation with the micelles of PCL -A:U-
25
PEG114, PCL -A:U-PEG , and PCL -A:U-PEG114, respec-
40
114
49
49
114
tively, at different concentrations (the total amount of PCL-A
and PEG-U in aqueous solution). The results demonstrate that
no obvious cytotoxicity against NIH/3T3 cells is observed even
the concentration of copolymer micelles is up to 1 mg/mL.
Therefore, these PCL-A:U-PEG micelles exhibit low cytotoxicity
to NIH/3T3 cells.
were destabilized and formed aggregates in response to pH 5.0
30
acetate buffer. Gao et al. reported that less than 20% DOX was
released from PCL-PEG micelles at pH 5.0 in one month.
Therefore, the fast release of DOX in acidic environment could
be attributed to the protonation of the amino group of nucleo-
base and shedding of micelle shell at acidic condition. This pH-
dependent releasing behavior is of particular interest in achieving
the tumor-targeted DOX delivery with micelles. Therefore,
micelles from supramolecular block copolymers may represent
a highly promising approach to achieve fast controlled drug
release.
Drug Loading and pH-Triggered Drug Release. To assess
the potential of supramolecular copolymer micelles based on
molecular recognition of uracil and adenine nucleobases, DOX
was used to evaluate the drug loading and release properties.
DOX is one of the most potent anticancer drugs in the treatment
of different types of solid malignant tumors and known to
interact with DNA by interaction and inhibition of macromole-
Cell Internalization. The cellular uptake of DOX-loaded
PCL -A:U-PEG micelles was evaluated by flow cytometry
49
114
4
5,46
cular biosynthesis.
It is well-known that physical entrapment
using HeLa cancer cells. After DOX-loaded micelles with DOX
concentration of 9 μg/mL were added to culture medium, the
HeLa cells were cultured at 37 °C for the predetermined time
intervals. Histograms of cell-associated DOX fluorescence for
HeLa cells are displayed in Figure 8. Remarkably, strong DOX
of hydrophobic drugs in polymeric micelles is mainly driven by
the hydrophobic interactions between the drug and the hydro-
30
phobic segments of polymers. Therefore, supramolecular block
copolymer PCL -A:U-PEG with the high PCL content was
4
9
114
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Figure 9. Confocal laser scanning microscopy (CLSM) images of HeLa cells that incubated with doxorubicin(DOX)-loaded PCL -A:U-PEG
114
4
9
micelles for (A) 30 min, (B) 6 h, and (C) 24 h. Cell nucleuses were stained with Hoechest 33342.
fluorescence intensity is observed in the cells after 5 min
incubation with DOX-loaded PCL -A:U-PEG micelles,
114
49
which is most likely due to significantly faster intracellular release
of DOX from PCL -A:U-PEG micelles. After a longer
49
114
incubation time of 3 h, the relative geometrical mean fluores-
cence intensities of DOX-loaded micelle pretreated cells are
about 50 fold of nonpretreated cells. The curves show that the
gradual increase intensity of fluorescence with increasing incuba-
tion time can be attributed to the cellular uptake of DOX-loaded
4
7,48
micelles.
The cellular uptake and intracellular release behaviors of
DOX-loaded micelles by HeLa cells were further investigated
by CLSM. HeLa cells were cultured at 37 °C for 30 min, 6 h, and
2
4 h, respectively, after DOX-loaded micelles were added to
culture medium with a DOX concentration of 9 μg/mL. Then
the nucleus was stained with Hochest33342 and the pretreated
cells were observed under CLSM. As shown in Figure 9, the
DOX fluorescence is observed mainly in the cytoplasm of the
cells when the cells are cultured in the DOX-loaded PCL -A:U-
Figure 10. Cell viability of HeLa cells against doxorubicin (DOX)-
loaded PCL -A:U-PEG micelles after cultured for 48 h with different
4
9
114
DOX doses.
49
properties of PCL-A:U-PEG micelles are very important for its
use as an ideal drug carrier.
PEG114 micelles for 30 min and 6 h. However, the fluorescence is
partly localized in the cell nucleus when cells are incubated with
the DOX-loaded micelles for 24 h. These results indicate that
DOX-loaded PCL -A:U-PEG micelles may be taken up by
In Vitro Cytotoxicity of Drug-loaded Micelles. The in vitro
cytotoxicity of DOX-loaded PCL -A:U-PEG copolymer mi-
49
114
4
9
114
celles compared with that of free DOX was evaluated by MTT
assay against HeLa cancer cells. The HeLa cells were treated with
the DOX-loaded PCL -A:U-PEG micelles and free DOX at
the cells through a nonspecific edocytosis mechanism and the
47,49
DOX molecules are released in endocytic compartments.
49
114
Therefore, the novel PCL-A:U-PEG micelles are not only
transported efficiently but also resided in cytoplasm. These
different DOX dose from 0.2 to 4 μg/mL. The HeLa cell
proliferation results are shown in Figure 10. It is found that the
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(
IC ) is 1.6 μg/mL. This result demonstrates that DOX-loaded
5
0
(
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4
9
114
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(
(
30,49
than the loaded drug.
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(
micelles can be attributed to the time-consuming DOX release
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capable of rapidly releasing DOX inside the cells to yield
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2
7
(
(
’
AUTHOR INFORMATION
(
1
Corresponding Author
(
*
Tel.: þ86-21-34205699. Fax: þ86-21-34205722. E-mail:
bshzhu@sjtu.edu.cn; xyzhu@sjtu.edu.cn.
(
(
’
ACKNOWLEDGMENT
(
This work was financially supported by the National Natural
Science Foundation of China (20974062, 30700175) and
National Basic Research Program 2009CB930400, Shanghai
Natural Science Foundation of the Science and Technology
Commission of Shanghai Municipal Government (No.
(
1
(
0
9ZR1415100), a Fundamental Key Project (No. YG2010-
(
MS92) of Shanghai Jiaotong University, Shanghai Leading
Academic Discipline Project (No. B202), and China National
Funds for Distinguished Young Scientists (21025417).
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