Synthesis and pH-responsive self-assembly behavior of a fluorescent amphiphilic triblock copolymer mPEG-b-PCL-b-PDMAEMA-g-PC for the controlled intracellular delivery of doxorubicin

Article abstract of DOI:10.1039/c6ra01504b

In this work, well-defined pH-responsive methoxy poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly[2-(dimethylamino)ethyl methacrylate]-g-7-propinyloxy coumarin triblock amphiphilic copolymers (mPEG-b-PCL-b-PDMAEMA-g-PC) were synthesized using a combination of atom transfer radical polymerization (ATRP), ring opening polymerization (ROP) and click chemistry. The chemical structures and compositions of these copolymers were characterized using Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (¹H NMR). The molecular weights of the copolymers were obtained using ¹H NMR spectroscopy and gel permeation chromatography (GPC) measurements. Subsequently, the polymers could self-assemble into micelles which were investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescence spectroscopy. The pH-responsive self-assembly behavior of these triblock copolymers in water were investigated at different pH values of 5 and 7.4 for controlled doxorubicin release, the results indicated that the release rate of DOX could be effectively controlled by altering the pH, DOX was sealed in neutral surroundings and DOX release was triggered in acidic surroundings. CCK-8 assays and confocal laser scanning microscopy (CLSM) against HeLa cells indicated that the micelles had no associated cytotoxicity, possessed good biodegradability and biocompatibility, and identified the location of the DOX in HeLa cells. The DOX-loaded micelles possessed high cytotoxicity to HeLa cells and exhibited inhibition of the proliferation of HeLa cells. Moreover, these flexible micelles with an on-off switched drug release may offer a promising pattern to deliver a wide variety of hydrophobic payloads to tumor cells for cancer therapy.

Full text of DOI:10.1039/c6ra01504b

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ARTICLE
Synthesis and pH-responsive self-assembly behavior of a fluorescent
amphiphilic triblock copolymer mPEG-b-PCL-b-PDMAEMA-g-PC for the
controlled intracellular delivery of doxorubicin
Received 00th January 20xx,
Accepted 00th January 20xx
Lei Lia, Beibei Lua, Qikui Fanb, Lulu Weia, Jianning Wua, Jun Houd, Xuhong Guoa, c, Zhiyong Liua*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this work,
a well-defined pH-responsive methoxy poly(ethylene glycol-block-poly(ε-
caprolactone)-block-poly[2-(dimethylamino) ethyl methacrylate]-g-7-propinyloxy coumarin
triblock amphiphilic copolymer (mPEG-b-PCL-b-PDMAEMA-g-PC) were synthesized by
combination of atom transfer radical polymerization (ATRP), ring opening polymerization (ROP)
and click chemistry. The chemical structures and compositions of these copolymers were
characterized by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic
resonance (1H NMR). The molecular weights of copolymers were obtained by 1H NMR spectra and
gel permeation chromatography (GPC) measurements. Subsequently, polymers could self-
assemble into micelles which were investigated by dynamic light scattering (DLS), transmission
electron microscopy (TEM), and fluorescence spectra. The pH-responsive self-assembly behavior
of these triblock copolymers in water were investigated at different pH value of 5 and 7.4 for
controlled doxorubicin release, which were indicated that the release rate of DOX could be
effectively controlled by altering pH, and sealed DOX in neutral surroundings and triggered DOX
release in acidic surroundings. CCK-8 assays and confocal laser scanning microscopy (CLSM)
against HeLa cells indicated that micelles had no associated cytotoxicity, possessed good
biodegradability and biocompatibility, identified the location of the DOX in HeLa cells. While DOX-
loaded micelles possessed high cytotoxicity to HeLa cells and exhibited inhibition of the
proliferation of HeLa cells. Moreover, these flexible micelles with an on-off switched drug release
may offer a promising pattern to deliver a wide variety of hydrophobic payloads to tumor cells for
cancer therapy.
directed toward engineering ‘smart’ materials that are able to
respond to change such as variations in temperature,9-11 pH,12,
1. Introduction
or light in their environment.14, 15 Moreover, some stimuli-
13
Currently, polymeric micelles as promising nanosized
antitumor drug carriers are being extensively studied,1-3 due to
their unique properties and structures, such as nanoscale size,
core-shell structure, relatively high stability and prolonged
circulation in the blood.4, 5 The ability of amphiphilic block
copolymers to self-assemble in selective solvents have been
studied widely.6-8 And much scientific effort have been
responsive polymers can make micelles responsive to pH
value. In contrast to other pH-sensitive chemotherapeutics
delivery system, micelles are spotlighted for the following
advantages: (i) high circulation half-life via avoiding non-
selective uptake by the reticuloendothelial system (RES), (ii)
enhanced solvent power to hydrophobic anticancer drugs, (iii)
utilize the enhanced permeability and retention (EPR) effect
for passive targeting.16-19 For example, cancer tissue is also
reported to be acidic extracellularly.20-22 Polymeric micelles
offer many unique advantages, such as passive accumulation
in tumors, prolong circulation time in blood and the enhanced
uptake by tumors.23-25 In particular, micelles form from block
copolymer consisting of poly(ethylene glycol) (PEG) and poly(ε-
27
caprolactone) (PCL) have drawn considerable interests.26,
The PEG with excellent biocompatibility forms the hydrophilic
corona in micelles, thereby it can reduce nonspecific adhesion
of polymeric micelles surfaces to blood components. PCL, a
hydrophobic polymer approved by the FDA for biomedical
applications, is biocompatible, biodegradable, and highly
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permeable to drugs and PCL serves as hydrophobic core in the defined amphiphilic PCL-b-[PGMA-g-PC]-b-P(PEGMA) with
micelles, which can be used to load water-insoluble drugs.22, 26, fluorescence units through ROP, RAFT and click chemistry, and
28,
29
Poly(2-(N, N-dimethylamino)ethyl methacrylate) micelles were used as a nano-reservoir for controlled release
(PDMAEMA) is a weak base with a pKa at about 7. Under the of DOX for bladder cancer therapy. Xu et al51 developed pH-
pKa, PDMAEMA is hydrophilic as its amine groups are sensitive PMs self-assembled from amphiphilic poly(ethylene
protonated. Above its pKa, PDMAEMA is hydrophobic as its glycol)-copoly(n-butyl methacrylate-ran-methacrylic acid) for
amine groups are deprotonated.30-33 The self-assembly of DOX release, simultaneously modifying micellar shell with
amphiphilic block copolymers usually requires that copolymers folate to improve the efficiency of cellular uptake by tumor
have well-defined structures and narrow polydispersities, cells.
whcih were usually reached by using the “living”/controlled
polymerization methods, such as atom-transfer radical mPEG-b-PCL-b-PDMAEMA-g-PC with fluorescence units via
In this study, we synthesized a well-defined pH-responsive
34
polymerization (ATRP),12, and ring-opening polymerization ROP, ATRP and click chemistry with mPEG-Br as macroinitiator,
(ROP),35 and reversible addition-fragmentation chain transfer ε-CL and DMAEMA as monomers. Incorporation of a block with
37
polymerization (RAFT).36,
Despite the versatility of CRP fluorescence units as a End-capping group in the shell
techniques, it is still a challenge to find feasible means to displayed fluorescent property as DOX carriers in vitro. The
introduce functional groups onto polymer chains. These self-assembly and pH responsive properties of the copolymer
functional polymers, for example, fluorescent groups labeled in water were investigated. The release investigation of DOX
polymers, biofunctionalized polymers and drug containing from micelles indicated that the release rate of the drug could
polymers, which could find wide applications in chemistry, be effectively controlled by altering the pH value. Meanwhile,
materials science and biomedical science fields.38-40Moreover, the cellular uptake and cytotoxicity test to HeLa cells were
a fluorescent coumarin unit could be labeled in vivo in order to performed.
detect DOX-loaded micelles. “Click” chemistry is a good choice
2. Experimental
2.1. Materials
to synthesize well-defined functional polymers. Chen et al41, 42
synthesized poly(vinyl acetate) with fluorescence via a
combination of RAFT/MADIX and “click” chemistry. Wang et
al43 prepared amphiphilic centipede-like brush copolymers
with PCL and poly(ethyl ethylene phosphate) side segments via
combination of ROP and click chemistry by one-pot syntheses
strategy. Ivonne L et al44 synthesized PDMAEMA-b-PCL-b-
PDMAEMA with different composition via ATRP. The
encapsulation ability of AmB depend on micelle dynamics and
the length of the hydrophilic segment. Mao et al34 synthesized
PLLA-b-PDMAEMA copolymers via ROP and ATRP. Zhu et al45
demonstrated that compared to stably shielded polyplexes of
PDMAEMA-PEG-PDMAEMA analogue, reversibly shielded DNA
polyplexesbased on low molecular weight bioreducible,
PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers have
excellent colloidal stability under physiological salt conditions.
Zhu et al27 prepared PEG45-b-PBO9-b-PCL61 triblock and PEG45-
b-PCL62 diblock copolymers for comparative studies of micelle
formation and relative stability in acidic media. You et al46
prepared two types of fluorescent PDI-cored star
polyelectrolytes, and unimolecular fluorescent micelles with a
perylenediimide core were sensitive to change in pH values.
Thomas K et al47 synthesized a set of PEG-PCL-lPEI triblock
copolymers, with decreasing hydrophilic/hydrophobic ratio a
transition from partly water-soluble micelle-like assemblies to
mainly water-insoluble particle-like precipitates was observed.
Dong et al48 synthesized PDMAEMA-g-PEG cationic hydrogel
nanoparticles by distillation-dispersion copolymerization of
methoxy mPEG and DMAEMA. It was found that the
PDMAEMA-g-PEG nanoparticles performed pH, ionic strength,
and thermosensitivity in water swelling behavior. Ni et al49
mPEG (Alfa Aesar, Mn = 1900 g/mol), ε-CL ( sigma-Aldrich ) was
distilled under reduced pressure after being treated with CaH2.
Tin(II)ethylhexanoate (Sn(Oct)2, Sigma), 2-Bromoisobutyryl bromide
(BIBB, Aldrich), N, N, N', N, 'N''-pentamethyldiethylenetriamine
(PMDETA) were purchased from Sigma-aldrich. Copper (I) chloride
(99.999 %, Alfa Aesar) were used without further purification.
Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing
Huafeng United Technology Co. Ltd. 2-(Dimethylamino)ethyl
methacrylate (DMAEMA 98%, Aldrich) was filtered over aluminum
oxide to remove the inhibitor (MEHQ) before being polymerized.
Tetrahydrofuran (THF) was refluxed over sodium and distilled twice
before use. Dimethylformamide (DMF) and triethylamine (TEA)
were dried by refluxing over CaH2 and distilled before use. 7-
hydroxy coumarin, propargyl bromide and sodium azide were
purchased from Aladdin Reagent Company and used without
further purification. Enhanced Cell Counting Kit-8 (CCK-8, Shanghai,
Beyotime Biotechnology). Copper (I) chloride (99.999%, Alfa Aesar)
were used without further purification. HeLa cells (Institute of cells,
CAS, Shanghai) were used as received. Dulbecco's modified eagle
medium (DMEM), fetal bovine serum (FBS), pancreatic enzymes
were obtained from biological industries. 4% Paraformaldehyde, 4’,
6-Diamidino-2-phenylindole (DAPI) and Triton X-100 were
purchased from Solarbio.
2. 2 Characterization
1H NMR data were obtained by Nuclear Magnetic
Resonance Spectroscopy (NMR) using a BrukerDMX-500 NMR
spectrometer with CDCl3 as solvent. Fourier Transform
Infrared Spectroscopy (FT-IR) analysis was measured by IR-
Affinity-1 Model spectrophotometer using KBr pellets. The
molecular weight and molecular weight distribution of
copolymers were
chromatography (GPC) using
permeation chromatograph and THF was used as eluent. X-ray
photoelectron spectroscopy (XPS) was performed using an Axis
Ultra spectrometer with a monochromatized Al-Kα X-ray as
prepared
a series of well-defined amphiphilic triblock
copolymers mPEG-b-PCL-b-PDMAEMA by a combination of
ROP and ATRP, which could self-assemble into micelles or
vesicles in PBS buffer solution, depending on the length of
PDMA in the copolymer. Agarose gel retardation assays
demonstrated that these cationic nanoparticles can effectively
condense plasmid DNA. Deng et al50 studied mPEG-b-PCL-b-
PDMAEMA nanoparticles as the codelivery vector of
hydrophobic drug and pDNA. Chen et al3 synthesized a well-
measured
by
gel
permeation
Viscotek TDA 302 gel
a
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excitation source (225 W). The fluorescence spectra were hexane, and dried under vacuum to a constant weight at
measured by Hitachi F-2500
fluorescence 40oC(yield: 50.6% and 52.3%).
spectrophotometer at an excitation wavelength of 300 nm. 2. 3. 4 mPEG-b-PCL-b-PDMAEMA with sodium azide
Dynamic light scattering (DLS) measurements were performed mPEG-b-PCL-b-PDMAEMA (1.5 g, 0.2 mmol) was dissolved in
a
by a BECKMAN COULTER Delasa Nano C particle analyzer at a DMF (15 mL). Sodium azide (0.225 g, 3.46 mmol) was added to
fixed angle of 165°. Before the light scattering measurements, this solution, and the mixture was stirred at 65oC for 48 h. The
the sample solutions were filtered three times by using final reaction solution was diluted with DMF and purified by
Millipore Teflon (Nylon) filters with a pore size of 0.45 μm. All dialysis against water to remove any traces of salts and
measurements were repeated three times, and the average unreacted reactants using a membrane with molecular weight
result were accepted as the final hydrodynamic diameter (Dh) cutoff of 7000 Da. The azide-containing polymers were
and zeta potential (mV). Samples for transmission electron collected by freeze-drying.
microscopy (TEM) images were taken on an H-600 2. 3. 5 Synthesis of 7-propinyloxy coumarin (PC)
transmission electron microscope (Hitachi, Japan) operating at
The synthesis procedure was carried out according to the
120 kV. Confocal laser scanning microscopy images (Zeiss reported mehods.3, 42 A mixture of 7-hydroxy coumarin (1.62 g,
CLSM510) images and fluorescence microscope (OLYMPUS U- 10 mmol) in acetone (25 mL), K2CO3 (1.38 g, 10 mmol), KI
RFL-T, Japan) were operated at the excitation wavelength of (0.083 g, 0.5 mmol) and propargyl bromide (1.2 mL, 15 mmol)
480 nm.
2.
was added to a flask, and the mixture was stirred 24 h at 80oC.
3
Synthesis of mPEG-b-PCL-b-PDMAEMA-g-PC with Then the reaction mixture was cooled to room temperature
and extracted with CH2Cl2. The combined organic extracts
were washed with water, dried over anhydrous MgSO4 and
fluorescence triblock amphiphilic copolymer
2. 3. 1 Synthesis of mPEG-b-PCL diblock copolymer
mPEG-b-PCL were synthesized by ROP with different feed evaporated to afford a crude product, which was purified by
ratios ε-CL using Sn(Oct)2 as catalyst. The typical procedure recrystallization from anhydrous ethanol to give a white solid
was as follows: mPEG (3.8 g, 2 mmol), ε-CL (6 g, 52.6 mmol), (77.8% yield).
Sn(Oct)2 (0.04 g, 0.1 mmol), and anhydrous toluene (50 mL) 2. 3. 6 Synthesis of mPEG-b-PCL-b-PDMAEMA-g-PC by click
were added into a fresh flamed and nitrogen purged round- chemistry
bottomed flask. The flask was then placed in a thermostatted
The synthetic pathway was shown in Scheme 1. Azide-
oil bath at 120oC for 24 h. After the polymerization, the containing polymers (1.0 g, 0.057 mmol), CuBr (0.151 g, 1.0
mixture was cooled to room temperature. The product was mmol), and PC (80 mg, 0.40 mmol) were purged with nitrogen
dissolved in CH2Cl2, and precipitated in methanol three times. to remove the dissolved oxygen.), PMDETA (0.337 g, 2 mmol)
Finally, the precipitate was dried under vacuum to a constant was added under nitrogen atmosphere. Then the ampoule
weight at 35oC. The different degree of polymerization (DP) of were sealed and stirred at 60oC in the absence of oxygen for
PCL were synthesised by ROP in the some way, which were 24 h. The reaction mixture was exposed to air, and the mixture
named MP1 and MP2(yield: 90.6% and 95.3%).
2. 3. 2 Synthesis of mPEG-b-PCL-Br initiator
were diluted by DMF, and purified by dialysis against water to
remove unreacted reactants using a membrane with molecular
Typically, mPEG-b-PCL (4 g, 0.2mmol) and triethylamine (3 mL) weight cutoff of 7000 Da. The resultant polymers mPEG-b-PCL-
were first added into a 100 mL dry flask and After 30 mL of b-PDMAEMA-g-PC were collected by freeze-drying, which
anhydrous CH2Cl2 was added to dissolve mPEG-b-PCL under were named MPDP1 and MPDP2(yield: 65.5% and 66.8%).
nitrogen atmosphere.The flask was placed in an ice/water 2. 4 Self-assembly of mPEG-b-PCL-b-PDMAEMA-g-PC in
bath. 2.0 mL of 2-bromoisobutyryl bromide was added into the aqueous solution
flask dropwise over 1 h, and the reaction mixture was stirred
Samples for UV-vis, DLS and TEM were prepared as follows:
48 h at 30oC. The precipitate was filtered off. Then, the filtrate mPEG-b-PCL-b-PDMAEMA-g-PC (20 mg) were dissolved in THF
was washed three times sequentially with an aqueous solution (4 mL) and subsequently, deionized water (1 mL) were added
of sodium bicarbonate and water. The product was further dropwise over a period of 30 min under high-speed
concentrated by a rotary evaporator, and precipitated three stirring at room temperature. After 4 h, 8 mL water was added
times in methanol, and dried under vacuum to a constant to quench the micellar assembly, subsequently dialyzed
weight at 35oC(yield: 72.2% and 74.5%).
2. 3. 3 Synthesis of mPEG-b-PCL-b-PDMAEMA by ATRP
(molecular weight cut-off: 3500 Da) against distilled water for
36 h, and micelles with different concentrations could be
Synthesis of mPEG-b-PCL-b-PDMAEMA: a series of mPEG-b- obtained by diluting with distilled water. During this dialysis
PCL-b-PDMAEMA were prepared by ATRP of DMAEMA using process, the hybridized copolymers self-assembled into
mPEG-b-PCL-Br as initiator and CuBr/PMDETA as catalyst. The micelles with PCL cores and mPEG, PDMAEMA shells, which
reaction procedures are shown in Scheme 1, which named were named MPDP1 and MPDP2.
MPD1 and MPD2.
The typical procedure was described below: mPEG-b-PCL-Br
2. 5 DOX encapsulation and release studies
100 mg of MPDP1/MPDP2 and 10 mg of DOX•HCl was
(0.2 g, 0.03 mmol), DMAEMA (2.0 g, 9.0 mmol), CuBr (0.151 g, dissolved in DMF (4 mL) separately, and the two solutions
1.0 mmol), PMDETA (0.337 g, 2 mmol), and THF (30 mL). The were mixed in a vial and stirred for 30 min, a 3-fold excess of
flask was degassed with three freeze-evacuate-thaw cycles. TEA were added in order to obtain DOX base. Then the mixture
Then, the polymerization was performed at 65oC for 12 h. were added dropwise into water (80 mL) using a syringe pump
After being cooled to room temperature, the reaction flask under high-speed stirring. The DOX-containing suspension was
was open to air, and the crude product was diluted with THF then equilibrated under stirring at room temperature for 4 h,
and passed through a neutral alumina column to remove the followed by thorough dialysis (molecular weight cut-off: 3500
copper catalysts. Finally it was precipitated thrice into cold Da) against deionized water for 2 days to remove unloaded
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DOX. The obtained DOX-loaded micelles were named D- represented the concentration of DOX in the nth sample. The in
MPDP1 and D-MPDP2, respectively. vitro release experiments were carried out in triplicate at each
The DOX loading content (DLC) and loading efficiency (DLE) pH and the reported results were the average values with
were determined by UV-vis spectrophotometry at 480 nm. To standard deviations.
determine the drug loading level, a small portion of DOX- 2. 6 Cytotoxicity test
loaded micelles was withdrawn and diluted with DMF to a
The cytotoxic effects of polymers, free DOX or DOX-loaded
volume ratio of 9/1 (DMF/H2O). The amount of DOX mPEG-b-PCL-b-PDMAEMA-g-PC micelles were evaluated
encapsulated was quantitatively determined by a UV-vis. The against HeLa cells by the standard XTT. To perform cytotoxicity
calibration curve used for drug loading characterization were assay, HeLa cells were seeded at a density of 5000 cells per
established by the intensity of DOX with different well on a 96-well plate and cultured for 24 h. The samples
concentrations in DMF/H2O (9/1, v/v) solutions. The DLC were were prepared at a series of desired concentrations. Every
defined as the weight ratios of entrapped DOX to that of the experimental well was treated with the samples for 24 h and
DOX-loaded micelles. The DLE of DOX was obtained as the others were added with fresh medium as control. After
weight ratios between DOX incorporated in assembled incubation for 24 h, CCK-8 were added into each well to
micelles and that used in fabrication.
dissolve the formazan by pipetting in and out several times.
The absorbance of each well was measured at a test
wavelength of 450 nm. The cell viability of samples were
calculated as follow:18, 52, 53
weight of loaded drug
DLC (wt%) =
DLE (wt%) =
×100
×100
weight of polymer
Atest − Ablank
Cell viability (%) =
×100
weight of loaded drug
weight of drug in feed
Acontrol − Ablank
Where Atest and Acontrol represented the intensity determined
for cells treated with different samples and for controlling
cells, respectively, and Ablank was the absorbance of wells
without cells.
The in vitro DOX release profiles from the MPDP1/MPDP2
assembled micelles were evaluated using buffer solutions with
pH values 5 and 7.4 in a dialysis bag (molecular weight cut-off:
3500). The whole bag were placed into 35 mL PBS or acetate
buffer and shaken (200 rpm.) at 37oC.
At specified time intervals 4 mL (Ve) samples were taken and
an equal volume of fresh buffer added to maintain the total
volume. The concentrations of DOX in different samples were
analyzed by UV-vis spectrophotometry at 480 nm. The
cumulative percent drug release (Er) was calculated using
2. 7. Intracellular release of DOX
Confocal laser scanning microscopy (CLSM) was used to
visualize the subcellular localization and intracellular release
behavior of DOX-loaded micelles and free DOX for various
lengths of time (0.5 h, 4 h and 24 h). First, the HeLa cells were
seeded in a glass base dish with a coverslip at a density of 5000
cells and cultured in DMEM supplemented with 10% FBS for 24
h. Then DOX-loaded micelles and free DOX was added, and
cells were cultured for 0.5 h, 4 h and 24 h in a humidified 5%
CO2-containing atmosphere. Finally, the location of
intracellular fluorescence was validated using a CLSM imaging
system (Zeiss CLSM510) at the excitation wavelength of 480
nm.
V
n−1 Ci +VoCn
∑
e
1
Eq. (1).
Er (%) =
×100
mDOX
Where mDOX represented the amount of DOX in the micelle,
V0 was the volume of the release medium (V0 = 70 mL), Ci
represented the concentration of DOX in the ith sample and Cn
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Scheme 1. Synthesis of mPEG-b-PCL-b-PDMAEMA-g-PC with fluorescence triblock amphiphilic copolymer
NMR (Fig. 2(B)) and FT-IR (Fig. 1(B)). Moreover, the success of
the “click” reaction could also be confirmed from FT-IR
spectroscopy from Fig. 1(C). It was clearly observed that the
absorption peak at 2103 cm-1 attributed to the azide group,
and disappeared after the click reaction in Fig.1 (D). All the
results demonstrated that the click reaction had been
successfully achieved.
3. Results and discussion
3. 1 Synthesis and characterization of polymers
The overall experiment was illustrated in Scheme 1. The
mPEG-b-PCL-b-PDMAEMA-g-PC were synthesized with
different hydrophobic segments by ROP, ATRP and click
reaction. The azide-terminated diblock copolymer prepared by
a nucleophilic substitution reaction between mPEG-b-PCL-b-
PDMAEMA-Br and NaN3. Subsequently, the obtained
azidofunctionalized copolymer were involved in “click”
chemistry with 7-propinyloxy coumarin to prepare fluorescent
polymers.
Fig. 2 The 1H NMR spectra of mPEG-b-PCL (B) and mPEG-b-PCL-Br
(A).
Fig. 2 showed a comparison of 1H NMR spectra of mPEG-b-PCL
and mPEG-b-PCL-Br with peak assignments in CDCl3. In Fig. 2, the
small peak at 3.396 ppm (peak a) and the sharp singlet at 3.650
ppm (peak b) were respectively corresponding to the protons of
CH3O- and -CH2CH2O- units of mPEG block, whereas the signals of -
CH2- units of PCL block could be found in peak c, d, e, and f at 2.319
ppm, 1.657 ppm, 1.394 ppm and 4.072 ppm. Moreover, as
observed in Fig 2(A), compared with the spectrum of mPEG-b-PCL,
the 1H NMR spectrum of mPEG-b-PCL-Br displayed new chemical
Fig. 1 FT-IR spectra of mPEG (A), mPEG-b-PCL (B), mPEG-b-PCL-b-
PDMAEMA-N3 (C) and mPEG-b-PCL-b-PDMAEMA-g-PC (D).
The FTIR spectra of the mPEG, mPEG-
b
-PDMAEMA-
-PCL, mPEG-
b
g-PC were
-PCL-
b-
PDMAEMA-N3 and mPEG-
b
-PCL-
b
shown in Fig. 1. The intensive absorption peak at 1730 cm-1 of
(B), (C) and (D) were assigned to the carbonyl band of PCL in
1
-PCL was confirmed by H
comparison with mPEG (A). mPEG-
b
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shifts attributed to the protons of -COC(CH3)2- at 1.940 ppm (peaks
g). The structural characteristics of mPEG-b-PCL-b-PDMAEMA had
been determined by 1H NMR analysis in Fig. 3. We could find the
characteristic signals of protons in PDMAEMA block in Fig. 3, the
protons of -CH2N- and -CH3 of PDMAEMA block were corresponding
to the peaks k and i at 2.602 ppm and 0.909 ppm, which could be
observed at 2.602 ppm and 0.959 ppm. Furthermore, the
overlapped signals at 4.076 ppm, 2.322 ppm, and 1.816 ppm
belonged to the protons of -COOCH2CH2-, -N(CH3)2 and
-
COOCH2CH2- in PDMAEMA block. Synthesis of 7-propinyloxy
coumarin (PC) were carried out similar to the procedure described
in previous publications in Fig. 4.3, 42 The resonance at 4.773 ppm
was the characteristic signal of the methylene protons of propargyl
group, while resonance of the alkenyl proton was observed at 2.602
ppm in Fig. 4.
Fig. 3 The 1H NMR spectrum of mPEG-b-PCL-b-PDMAEMA (MPD2).
The obtained azido-functionalized copolymer were subsequently
involved in “click” chemistry with 7-propinyloxy coumarin to
prepare fluorescence copolymer. The copper (I) and its ligands were
reported to be very efficient to catalyze the 1, 3-dipolar
cycloaddition of organic azides with terminal alkynes. Herein,
CuBr/PMDETA are used as the catalytic system,54, 55 and DMF as
solvent for the 1, 3-dipolar cycloaddition of azido-functionalized
1
copolymer and 7-propinyloxy coumarin. Fig. 5 showed the H NMR
spectrum recorded for mPEG-b-PCL-b-PDMAEMA-g-PC. The
characteristic signals at around 7.680 ppm, 7.407 ppm, 6.963 ppm
and 6.315 ppm were assigned to the protons of the coumarin group
and the signals of alkenyl group of PC units at 2.602 ppm
disappeared completely, which further confirmed the formation of
mPEG-b-PCL-b-PDMAEMA-g-PC.
Fig. 4 The 1H NMR spectrum of PC.
The degrees of polymerization (DP) for the PCL could be
calculated about from the integration ratio between the methylene
protons (2.32 ppm) in the repeat units and those (3.70 ppm) in the
terminal unit based on 1H NMR spectrum.
The molecular weights (Mn, NMR) of MP1, MPD1, MP2 and MPD2
1
were calculated according to the H NMR analysis by the following
equations:
A
Ac
b
=
(1)
4× 43 2× NCL
Af + j
A
b
=
(2)
4× 43 2× N + NDMAEMA
(
)
CL
Fig. 5 The 1H NMR spectrum of mPEG-b-PCL-b-PDMAEMA-g-PC
Table 1 Composition of mPEG-b-PCL-b-PDMAEMA-g-PC block
copolymers
The number-average molecular weight (Mn) of the copolymer was
obtained by the following formula:
Mn of copolymer (MP1 and MP2) = 1900 + 114.14 × NCL;
Mn of copolymer (MPD1 and MPD2) = 1900 + 114.14 × NCL + 157.2 ×
NDMAEMA
a
b
c
c
Samples
Mn,Th
Mn,NMR
Mn,GPC
Mw/Mn
Where Ab, Ac and Af+j were the integral values of the peaks b, c and
f, j in Fig. 2 respectively; 114.14 was the molecular weight of one
repeating unit of the PCL block, 157.2 was the molecular weight of
the DMAEMA, and 43 is the polymerization degree of mPEG.
MP1
MPD1
MPDP1
MP2
7942
10035
17940
——
8990
15980
16567
12757
23250
25258
1.29
1.13
1.15
1.09
1.16
1.18
16320
——
10450
18327
——
13440
19482
——
MPD2
MPDP2
aCalculated by theory analysis from the feed ratio of monomers to
1
initiator; bCalculated from H NMR data; cPolymerization conditions
[monomer]0/[mPEG-b-PCL-Br]0/[CuBr]0/[PMDETA]0=100/1/1/2,
6 | J. Name., 2012, 00, 1-3
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1
measured by GPC calibrated with PS standards. THF was used as theoretical values and H NMR analysis, which were suggested that
eluent.
MPDP block copolymers were synthesized and characterized
successfully.
3. 2. Formation and Characterization of the blank and DOX-loaded
mPEG-b-PCL-b-PDMAEMA-g-PC (MPDP1 and MPDP2) block
copolymers micelles
As an amphiphilic block copolymers, when the concentration
were above the critical micelle concentration (CMC), mPEG-b-PCL-
b-PDMAEMA-g-PC could self-assemble into micelles in selective
solvent. The hydrophilic mPEG and PDMAEMA chains were mainly
in the corona of the micelles, whereas the hydrophobic PCL side
chains were mainly in the core of the micelles. The hydrophobic of
PCL as cores had been extensively used for drug delivery system.
DOX was physically incorporated into MPDP1 and MPDP2 polymeric
micelles, which were named D-MPDP1 and D-MPDP2. The physico-
chemical properties of the blank and DOX-loaded micelles were
characterized by DLS analysis, respectively. Hydrodynamic diameter
(Dh), polydispersity index (PDI), zeta potentials of the blank and
DOX-loaded micelles were summarized in Table 2.
Fig. 6 Evolution of GPC chromatograms of mPEG-b-PCL, mPEG-
b-PCL-b-PDMAEMA and mPEG-b-PCL-b-PDMAEMA-g-PC block
copolymers.
The GPC traces of MP1, MPD1, MPDP1, MP2, MPD2 and MPDP2
block copolymers were shown in Fig. 6. All the curves of MPD1,
MPDP1 and MPD2, MPDP2 shifted to lower elution time compared
to that of MP1 and MP2. The detailed information about the
molecular weights and PDI of the triblock copolymers were listed in
Table 1. The GPC results were almost consistent with the
Table 2 Hydrodynamic diameter (Dh), size distributions (PDI) and
zeta potentials of blank and DOX-loaded mPEG-b-PCL-b-PDMAEMA-
g-PCmicelles.
Blank
DOX-load
Micelle
DLC
DLE
Dh (nm)
PDI
Zeta (mV)
Dh (nm)
PDI
Zeta (mV)
(wt%)
(wt%)
MPDP1
MPDP2
78±1.3
95±2.2
0.280±0.035
0.156±0.021
24.67±0.32
26.05±0.24
102±2.5
115±3.1
0.135±0.073
0.216±0.035
2.50±0.22
5.35±0.45
4.58
5.43
45.8
54.3
Fig. 7 The particle size distribution curves corresponding to the
samples in MPDP1 and D-MPDP1 (A), MPDP2 and D-MPDP2 (B) and
zeta potentials of DOX-loaded polymeric micelles in MPDP1 and D-
MPDP1 (C), MPDP2 and D-MPDP2 (D).
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Fig. 8 TEM images of mPEG-b-PCL-b-PDMAEMA-g-PC polymeric
Fig. 10 (a) UV-vis spectrum of MPDP1 micelles and (b) fluorescence
micelles (MPDP1 (A, B) and MPDP2 (C, D)).
spectra of mPEG-b-PCL-b-PDMAEMA-g-PC micelles solution.
Fig. 9 Plots of transmittance as a function of temperature for mPEG-
b-PCL-b-PDMAEMA-g-PC copolymer micelle solution (A), particle
size of the mPEG-b-PCL-b-PDMAEMA-g-PC in
H2O at room
temperature (B).
As shown in Fig. 7. The Dh, PDI and zeta potential of the
micelles were evaluated by DLS and TEM. The combination of TEM
and DLS data showed a spherical morphology with the respective
average radii around 78 and 95 nm, respectively(Fig. 7, and Table
2). In contrast, Dh were measured by TEM in Fig 8, respectively, the
smaller size were probably due to the difference in the two
measuring methods. The dehydration of the micellar shell during
the TEM samples preparation process may have led to a decrease in
the size of the micelles. Further, discrepancies were expected
because DLS, reported an intensity-average diameter, whereas TEM
Fig. 11 Fluorescence images of PC (A), MPDP1 (B)， MPDP2 (C)，D-
MPDP1 (D) and D-MPDP2 (E) micelles. PC (blue), DOX (red).
The UV-vis spectrum of MPDP1 micelles showed a characteristic
UV-vis absorption peak of coumarin moiety at 300 nm in Fig. 10 (A).
The fluorescence spectra of mPEG-b-PCL-b-PDMAEMA-g-PC were
displayed in Fig. 10(B). It could be observed that the triblock
copolymer, obtained by coupling of azide-containing polymers with
7-propinyloxy coumarin, exhibited a strong fluorescence peak at
about 425 nm. Fig. 11 showed fluorescence images of PC (A),
MPDP1 (B)， MPDP2 (C)，D-MPDP1 (D) and D-MPDP2 (E) micelles.
We could see bright blue of PC, MPDP1 and MPDP2 in Fig.11(A, B
and C). The DOX-loaded micelles displayed the bright blue
fluorescence corresponding to the coumarin dye implying that the
fluorescent micelles had been effectively internalized.
reported a number-average diameter. Thus, for
a given size
distribution of finite polydispersity, TEM images will always
undersize relative to DLS data. When DOX was loaded into the
polymeric micelles in the core and adsorbed drug on the surface. It
could also be found that the zeta potentials of the drug-loaded
micelles were lower than those of blank micelles for both of the
polymers, resulting from decreased charge density because of
larger particle sizes in the Table 2 and Fig. 7. As shown in Fig. 9, a
stability assay in terms of transmittance and particle size of mPEG-
b-PCL-b-PDMAEMA-g-PC micelles were investigated in water for (A)
and (B). The MPDP1 (A) micelles presented reversible
transformation of transparency and turbidity during the reversible
cooling and heating cycles, when the temperature increases,
hydrogen bonding could not be weakened but micelles start to
contract because of the decrease in electrostatic repulsion between
PDMAEMA chains, indicating that the coronas are hydrophobic to
some extent. Obviously, the phase transition of the micelles were
reversible, which indicated that the mPEG-b-PCL-b-PDMAEMA-g-PC
micelles were stable. As shown in Fig. 9(B), no obvious change of
the particle size of the D-MPDP1 and D-MPDP2 micelles during 50 h
indicated that the micelles had a well long-term stability without
the presence of precipitation and phase separation. The result
revealed that the MPDP1 and MPDP2 micelles could offer the
protection of drugs from untimely structure disintegration and
premature drug release until arriving a disease site.
3. 3 In vitro release of DOX from micelles
Fig. 12 In vitro release of DOX from various DOX-loaded polymeric
micelles at 37 °C under different pH conditions.
As expected, the mPEG-b-PCL-b-PDMAEMA-g-PC micelles
exhibited a pH-responsive characteristic. In vitro drug release
performances of the micelles were performed under physiological
conditions (PBS, pH= 5 and 7.4) at 37oC in Fig. 12. It could be
observed that the drug release rates of DOX from micelles were
obviously changed by pH values as well as time. With regard to pH
of 7.4 at 37oC, the micelles stayed compact and the loaded DOX was
released slowly. After 5 h, less than 20% of DOX (8.6% and 15.1%
for D-MPDP1 and D-MPDP2, respectively) were released. Even after
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24 h, only about 26.1% and 35.1% for D-MPDP1 and D-MPDP2 were
released, respectively. In contrast, when the pH was lower at 37oC
(pH=5), the drug release were accelerated. After 24 h, the
cumulative release were 83.1% and 88.7% for D-MPDP1 and D-
MPDP2, respectively. Although the mechanism of drug release from
polymeric matrices was very complex and was still not completely
understood, it could be simplistically classified as either pure
diffusion, erosion controlled release or a combination of the two
mechanisms.56, 57 In this study, the results were due to the swollen
drug-loaded micelles, attributing to the protonation of amino
groups in PDEAEMA segments at weakly acidic conditions.
Furthermore, the accelerated micelles rate might be related to the
swollen hydrophobic core, which could cause the drug molecules
close to the surface to diffuse into the medium. Meanwhile, the
DOX molecules were not only encapsulated inside the micellar core,
but also absorbed by the PDMAEMA shell due to the electric action,
while only that loaded by hydrophobic effect could be released
comparatively fast, so it might spend extended period to achieve
complete release. These MPDP1 and MPDP2 micelles were just like
on-off switching nanocarriers in release kinetics by changing pH
values. Therefore, it were highly interesting for intracellular anti-
cancer drug delivery.
Fig. 14 Cell viability of HeLa cells incubated with free DOX, DOX-
loaded micelles (D-MPDP1 and D-MPDP2) for 48 h at different
concentrations.
Fig. 14 showed the results of samples treated with free DOX and
DOX-loaded micelles for 48 h, respectively. The DOX dosages
required for the inhibitory concentration to produce 50% cell death
(IC50) were 0.633 μg/mL, 1.208 μg/mL, 1.092 μg/mL for 48 h for free
DOX, D-MPDP1 and D-MPDP2 against HeLa cells, this slight
difference between two DOX-loaded micelles could be explained
that the latter containing more PCL segments, leading to higher
drug loading level and more sensitive, respectively. Both of the DOX
3. 4 Cytotoxicity test
Cytotoxic effects of the polymers, free DOX or DOX-loaded loaded micelles showed slightly lower cytotoxicity than free DOX
micelles in HeLa cells were determined by CCK-8 assay. The cell due to the time-consuming DOX release from micelles in
viability of blank micelles and DOX-loaded micelles against HeLa comparison to free DOX at the same concentration. All of DOX-
cells were evaluated. The cell viability of blank micelles were loaded micelles had a similar capacity of killing tumor cells as free
measured after 48 h incubation. The blank micelles with different DOX for 48 h, which were indicated that DOX-loaded micelles might
concentration were nontoxic to HeLa cells and the cell viability were not inhibit the ability of killing cells although it slowed down the
over 95% at all concentrations (12.5~200 μg/mL) tested in Fig. 13, release of DOX. Moreover, the results revealed that DOX released
from the micelles could exploit a potent drug efficacy as free DOX
after entry into the HeLa cells. It could produce the desired
pharmacological action and minimize the side effect of free DOX.
which were indicated that the blank micelles could be used as a
delivery system for anticancer agents.
3. 5 In vitro cellular uptake studies
Fig 13 In vitro cell viability of the mPEG-b-PCL-b-PDMAEMA-g-PC
micelles. Concentration-dependent cell viability of HeLa cells
treated with the MPDP1 (A) and MPDP2 (B) after incubation of 48 h.
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Scheme 2. Illustration of pH-responsive self-assembly of mPEG-b-
PCL-b-PDMAEMA-g-PC copolymer for the efficient intracellular
release of anti-cancer drugs triggered by the acidic
microenvironment inside the tumor tissue.
4. Conclusion
In the current work, we had reported pH-responsive mPEG-
-PCL-b-PDMAEMA-g-PC copolymers by click chemistry, ROP,
b
ATRP with fluorescence units for cancer therapy. The
Hydrodynamic diameter, polydispersity index (PDI) and zeta
potential of the mPEG-b-PCL-b-PDMAEMA-g-PC micelles were
evaluated by DLS and TEM, and the sizes of MPDP1 and
MPDP2 micelles with spherical shapes determined by TEM
were about 78 nm and 95 nm. These copolymers could
markedly improve micellar stability and extend the range of
applications of micelles in controlling drug delivery with
increasing PCL segments. In addition, DOX-loaded micelles had
a fluorescence, which were investigated by fluorescence
spectrophotometer and fluorescence microscope in order to
label the location of drug in vivo.
The release behaviors of DOX from micelles exhibited pH-
responsive in vitro, the DOX loading contents were the higher
as the PCL segment increased. And the release of DOX from
the micelles were significantly accelerated by decreasing pH
from 7.4 to 5.0 at 37oC. After 50 h for D-MPDP2 micelles, the
cumulative release were about 88.7% (w/w), which could be
provided sustained drug delivery behavior after the DOX-
loaded micelles entered into blood circulation by endocytosis.
In addition, the cytotoxicity tests revealed that these MPDP
micelles possessed good biodegradability and biocompatibility
for the HeLa cells and the DOX-loaded micelles showed much
higher toxic effect, which were almost similar to free DOX.
Moreover, the DOX loaded micelles were taken up by the
nuclei of cells via the indocytosis process by CLSM.
Furthermore, the applicability of these micelles toward tumor-
targeting delivery applications in vivo is highly promising
chemotherapy in the future.
Fig. 15 Confocal laser scanning microscopy images of HeLa cells
incubated with (A) free DOX and (B) DOX-loaded (D-MPDP2) for
different times. The DOX dosage was 10 μg/mL. For each panel,
images from left to right show cell nuclei stained by DOX
fluorescence in cells (red), bright field of cells, HeLa (blue), and
overlays of the blue and red images. The scale bars are 20 μm in all
images.
To evaluate the intracellular uptake efficiency, confocal laser
scanning microscopy were used to identify the location of the
DOX in HeLa cells. The CLSM of HeLa cells after 0.5, 4 and 24 h
of incubation with free DOX and DOX-loaded micelles (D-
MPDP2) were presented in Fig. 15. After incubation for 0.5 h,
stained with DAPI, the nuclei and cytoplasm of pretreated cells
were observed by CLSM. By comparison with the control in Fig.
15(B), the observation revealed that free DOX was slightly
accumulated in the cell nuclei of HeLa cells in Fig. 15(A). After
incubation for 4 h and 24 h, free DOX was larger accumulated
than DOX-loaded micelles (D-MPDP2). While DOX released
from D-MPDP2 were mainly located in the cytoplasm, and DOX
was released into the cytoplasm and nuclei of cells under acid
conditions in lysosomes in Fig. 15(A and B). The result also
indicated that free DOX was taken up by diffusion through the
cell membrane and the DOX loaded micelles were taken up by
the nuclei of cells via the indocytosis process. Moreover, the
self-assembled micelles showed a great potential as anti-
tumor drug carriers for cancer therapy.
Acknowledgements
The authors gratefully acknowledge financial supports from the
National Natural Science Foundation of China (21367022) and
Bingtuan Innovation Team in Key Areas (2015BD003).
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DOI: 10.1039/C6RA01504B
Schematic illustration of pH-responsive self-assembly of mPEG-b-PCL-b-PDMAEMA-g-PC copolymer with a
fluorescent coumarin units for controlling the DOX release