Well-defined thermoresponsive dendritic polyamide/poly(N-vinylcaprolactam) block copolymers

Article abstract of DOI:10.1002/pola.26716

The first- and second-generation well-defined thermoresponsive amphiphilic linear-dendritic diblock copolymers based on hydrophilic linear poly(N-vinylcaprolactam) and hydrophobic dendritic aromatic polyamide have been synthesized via reversible addition fragmentation chain transfer polymerization of N-vinylcaprolactam by employing dendritic chain-transfer agents possessing a single dithiocarbamate moiety at the focal point. These linear-dendritic copolymers exhibit reversible temperature-dependent phase transition behaviors in aqueous solution as characterized by turbidity measurements using UV-vis spectroscopy. Their lower critical solution temperatures depend on the generation of the dendritic aromatic polyamides and the concentrations of the copolymer solutions. These amphiphilic copolymers are able to form nanospherical micelles in the aqueous solution as revealed by fluorescent spectroscopy, dynamic light scattering, and transmission electron microscope (TEM). The core-shell structure of micelles has been proved by ¹H NMR analyses of the micelles in D2O. The micelles loaded with indomethacin as a model drug showed high-drug loading capacity and thermoresponsive drug release behavior.

Full text of DOI:10.1002/pola.26716

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Well-Defined Thermoresponsive Dendritic Polyamide/
Poly(N-vinylcaprolactam) Block Copolymers
Yunmei Bi, Caixian Yan, Lidong Shao, Yufei Wang, Yongcui Ma, Gang Tang
Department of Medicinal Chemistry, College of Chemistry and Chemical Engineering, Yunnan Normal University,
Kunming 650092, People’s Republic of China
Correspondence to: Y. Bi (E-mail: yunmeibi@hotmail.com)
Received 23 January 2013; accepted 3 April 2013; published online 00 Month 2013
DOI: 10.1002/pola.26716
ABSTRACT: The first- and second-generation well-defined ther-
moresponsive amphiphilic linear–dendritic diblock copolymers
based on hydrophilic linear poly(N-vinylcaprolactam) and
hydrophobic dendritic aromatic polyamide have been synthe-
sized via reversible addition fragmentation chain transfer poly-
merization of N-vinylcaprolactam by employing dendritic
chain-transfer agents possessing a single dithiocarbamate moi-
ety at the focal point. These linear–dendritic copolymers exhibit
reversible temperature-dependent phase transition behaviors
in aqueous solution as characterized by turbidity measure-
ments using UV–vis spectroscopy. Their lower critical solution
temperatures depend on the generation of the dendritic aro-
matic polyamides and the concentrations of the copolymer
solutions. These amphiphilic copolymers are able to form
nanospherical micelles in the aqueous solution as revealed by
fluorescent spectroscopy, dynamic light scattering, and trans-
mission electron microscope (TEM). The core–shell structure of
micelles has been proved by 1H NMR analyses of the micelles
in D2O. The micelles loaded with indomethacin as a model
drug showed high-drug loading capacity and thermorespon-
C
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sive drug release behavior.
2013 Wiley Periodicals, Inc. J.
Polym. Sci., Part A: Polym. Chem. 2013, 00, 000–000
KEYWORDS: controlled drug release; dendritic aromatic polyam-
ide; diblock copolymers; micelles; poly(N-vinylcaprolactam); re-
versible addition fragmentation chain transfer (RAFT)
Thermoresponsive polymers have attracted considerable
interests in the last years owing to their unique properties.
They undergo a reversible phase transition at lower critical
solution temperature (LCST). It has been known that poly-
mer architecture affects the thermal phase transition behav-
ior of thermoresponsive polymer solutions.17–21 Although
many efforts have been made to impart the thermorespon-
sive character into dendritic polymers, until recently, only a
few studies related to thermoresponsive linear–dendritic
block copolymers have been reported. Among them,
thermoresponsive poly(N-isopropylacrylamide), polyethylene
glycol (PEG), and poly(2-isopropyl-2-oxazoline) linear chains
have been attached to poly(benzyl ether) dendrons, aliphatic
polyester or poly(L-lysine) to obtain thermoresponsive
linear–dendritic block copolymers.22–26
INTRODUCTION Linear–dendritic block copolymers consist of
covalently bound linear and dendritic segments. Because of
combining high functionality of dendrimers and processabil-
ity of linear polymers, they are promising for many applica-
tions, including controlled drug delivery, catalysis, and the
biomineralization of calcium carbonate.1–8 Amphiphilic lin-
ear–dendritic copolymers are one of the most important
types of linear–dendritic copolymers. Compared with con-
ventional amphiphilic block copolymers, they possess unique
solution and bulk properties because of the nonentangled,
but densely packed structure of the dendritic segment.9,10
The unique amphiphilic character of the linear–dendritic
block copolymers with hydrophobic dendritic block and
hydrophilic linear block enable them to self-assemble to
form versatile micelles in an aqueous phase. The dendritic
block of the copolymer forms the core of the micelle,
whereas the hydrophilic flexible chain forms a protective
corona around the micelle. The micelles are able to encapsu-
late a large number of small guest molecules not only in the
internal voids of the dendrons, but also in the space
between individual monodendrons. Therefore, these amphi-
philic linear–dendritic block copolymer systems have been
However, little attention has been paid to poly(N-vinylcapro-
lactam) (PNVCL)-containing linear–dendritic block copoly-
mers. PNVCL is a typical thermoresponsive polymer derived
from an inexpensive commercially available monomer, N-
vinylcaprolactam (NVCL).27 It undergoes a phase transition
in aqueous solution at temperatures close to physiological
temperatures (32–38 ꢀC). It is also nonionic, nontoxic, and
biocompatible.28,29 Furthermore, different from most
proposed
for drug
delivery and
gene
therapy
applications.11–16
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polyacrylamides, its hydrolysis does not produce amine com-
pounds. These properties make PNVCL a suitable polymer
for use in biomedical applications in vivo.30–34 But NVCL is
one of the unconjugated monomers whose polymers can be
prepared only via radical polymerization. The living=con-
trolled radical polymerization of unconjugated monomers
represented a synthetic challenge owing to the highly reac-
tive radical species derived from them and the lack of suita-
measured by gel permeation chromatography=multiangle
laser light scattering (GPC=MALLS). The GPC-MALLS system
consisted of a Waters 2690D separations module and a
Waters 2414 refractive index detector (RI), and a Wyatt
DAWN EOS MALLS detector. Styragel HR1 and HR4 columns
(Waters) were used at 40 ꢀC with polystyrene as standard
and THF as a mobile phase at a flow rate of 0.3 mL=min.
ble reagents or catalysts that can induce
a
fast
Synthesis
interconversion between the highly reactive radical and the
dormant species. But situation has been improved these
years, and controlled radical polymerization of NVCL via re-
versible addition fragmentation chain transfer (RAFT) poly-
merization or macromolecular design via the interchange of
xanthate has been reported.35–38 Atom-transfer radical poly-
merization and cobalt-mediated radical polymerization of
NVCL have also been developed.39–43 A few well-defined
thermoresponsive linear PNVCL-containing block copolymers
have been synthesized.44,45
Synthesis of G1-COOH (3,5-Bis(benzoyl-amino)
benzoic Acid)
G1-COOH was synthesized according to the previously pub-
lished literature47 with modification. To a solution of 3,5-dia-
minobenzoic acid (3.04 g, 20 mmol) in 40 mL of NMP,
benzoyl chloride (6.8 mL, 58.6 mmol) was added dropwise
ꢀ
at 0 C and stirred for 3 h. The reaction mixture was poured
into HCl=water (v=v 5 1:20), and the precipitate was col-
lected by filtration, washꢀed with methanol for three times,
and dried in vacuo at 45 C to give a grayish solid. The yield
was 98.6 % (7.10 g).
In this report, we synthesized first- and second-generation
well-defined linear–dendritic block copolymers containing
PNVCL chains and polyamide dendrons via RAFT polymeriza-
tion of NVCL by using dendritic chain-transfer agents (CTAs)
possessing a single dithiocarbamate moiety at the focal point.
To the best of our knowledge, this is the first report of the
syntheses of PNVCL-containing linear–dendritic block copoly-
mers. The thermoresponsive and micellar behaviors of these
block copolymers were investigated. The controlled release
of indomethacin (IMC) model drug using the thermorespon-
sive micelles was also studied.
1H NMR (500 MHz, DMSO-d6, d): 12.98 (br, 1H, COOH),
10.49 (s, 2H, CONH), 8.65 (s, 1H, CH), 8.16 (d, 2H, J 5 5 Hz,
H, CH), 7.94–8.01 (m, 4H, Ar H), 7.49–7.64 (m, 6H, Ar H).
Synthesis of G1-COOCH3 (Methyl 3,5-bis
(benzoylamino)benzoate)
To a solution of G1-COOH (1.50 g, 4.2 mmol) in 100 mL of
methanol, sulfuric acid (20 mL) was added dropwise at 66–
67 ꢀC. After the addition was completed, the reaction mix-
ture was refluxed for 1.5 h. After cooling to room tempera-
ture, the reaction mixture was neutralized with saturated
Na2CO3 aqueous solution. After removing the solvents by a
rotary evaporator, the precipitate was collected by filtration.
Recrystallization from hot water afforded a grayish solid.
The yield was 80.3 % (1.26 g).
EXPERIMENTAL
Materials
NVCL (98%, Aldrich) was distilled under reduced pressure
and then stored at 4 ꢀC. a,a-Azobisisobutyronitrile (AIBN)
(98%, Sinopharm Chemical Reagent) was purified by recrys-
tallizing from methanol two times. N-methyl-2-pyrrolidone
(NMP) (Sinopharm Chemical Reagent) was dried over 4 Å
molecular sieves and distilled under reduced pressure just
prior to use. Tetrahydrofuran (THF) was refluxed on sodium
and distilled from sodium benzophenone. N,N-diethyl-S-(a,a0-
dimethyl-a00-actetic acid) dithiocarbamate was prepared
according to the method described in the previously pub-
lished literature.46 3,5-Diaminobenzoic acid, benzoyl chloride,
sodium borohydride (NaBH4, 98%), 4-dimethylamino-pyri-
dine (DMAP, 99%) were obtained from Sinopharm Chemical
Reagent without further purification. N,N0-dicyclohexylcarbo-
diimide (DCC, 99%, Acros Organics) and other reagent were
used as received.
1H NMR (500 MHz, CDCl3, d): 8.48 (s, 1H, Ar H), 8.05–8.10
(d, J 57.35 Hz, 2H, Ar H), 7.88–7.90 (m, 4H, Ar H), 7.52–
7.59 (m, 6H, Ar H), 3.94 (s, 3H, CH3). 13C NMR (125 MHz,
CDCl3, d): 166.53, 166.21, 139.54, 134.71, 131.51, 130.43,
128.20, 127.71, 117.12, 113.16, 52.03. IR (KBr): m 5 3460
(NAH), 2925, 2855 (CAH), 1633 (C@O), 1580, 1428 (Ar),
1267, 1195 (CAO), 841, 713 cm21 (Ar). ESIMS (m=z (%)):
375 [M 1 H]1.
Synthesis of G1-CH2OH (3,5-Bis(benzoylamino)
benzyl alcohol)
To a solution of G1-COOCH3 (0.5 g, 1.3 mmol) in 20 mL of
THF, NaBH4 powder (0.2 g, 5.3 mmol) was added. The result-
ing suspension was stirred at 65 ꢀC for 15 min. Methanol
(20 mL) was then added dropwise and the reaction mixture
was refluxed for 7 h. After cooling to room temperature, the
reaction mixture was quenched with 2M HCl (40 mL). The
organic layer was separated and the aqueous phase
extracted with ethyl acetate (3 3 30 mL). The combined or-
ganic phase was dried over anhydrous magnesium sulfate,
Characterization
1H and 13C NMR spectra were obtained on a Bruker DRX-
500 spectrometer. ESI-MS spectra were recorded on a Finni-
gan LCQ-Advantage mass spectrometer. FTIR spectra were
measured on a Nicolet AVATAR 360 FTIR spectrometer. Mo-
lecular weight and polydispersity of polymers were
filtered, and evaporated to obtain
a
solid residue.
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Recrystallization from ethanol afforded the alcohol (G1-OH)
as grayish powder. The yield was 95.6 % (0.43 g).
room temperature. The reaction mixture was poured into
HCl (1M), and the precipitate was collected by filtration,
washed with methanol for three times, and dried in vacuo at
1H NMR (500 MHz, DMSO-d6, d): 10.29 (s, 2H, CONH), 8.21 (s,
1H, Ar H), 7.98–7.99 (m, 4H, Ar H), 7.51–7.59 (m, 8H, Ar H),
4.51 (s, 2H, CH2). 13C NMR (125 MHz, DMSO-d6, d): 165.91,
143.54, 141.48, 139.45, 135.31, 131.89, 128.71, 128.02,
115.01, 114.70, 112.31, 63.40. IR (KBr): 3414 (NAH), 2924,
2848 (CAH), 1622 (C@O), 1429 (Ar), 1270, 1195 (CAO), 841,
713 cm21 (Ar). ESIMS (m=z (%)): 347 [M1H]1.
ꢀ
45 C to give a grayish solid. The yield was 89.0 % (3.57 g).
1H NMR (500 MHz, DMSO-d6, d): 13.0 (br, 1H, COOH), 10.50
(s, 6H, CONH), 8.66 (s, 3H, Ar H), 7.97–8.17 (m, 14H, Ar H),
7.54–7.63 (m, 12H, Ar H).
Synthesis of G2-COOCH3
G2-COOCH3 was synthesized by the same general procedure
described for the synthesis of G1-COOCH3. From G2-COOH
(1.50 g, 1.8 mmol), methanol (200 mL) and sulfuric acid (22
mL) were obtained 1.23 g (80.4 %) as a grayish solid.
Synthesis of the [G-1]-based RAFT Agent
A solution of G1-OH (0.37 g, 1.1 mmol), N,N-diethyl-S-(a,a0-di-
methyl-a00-actetic acid) dithiocarbamate (0.29 g, 1.2 mmol),
and DMAP (0.035 mg) in dry THF (20 mL) was cooled in
ice-water bath, and then DCC (0.26 g, 1.2 mmol) was added.
After stirring in ice-water bath for 1 h, the mixture was
stirred for another 8 h at room temperature. After removing
N,N-dicyclohexylurea formed by suction filtration, the filtrate
was concentrated and further purified by silica gel column
chromatography using petroleum ether=ethyl acetate (v=v 5
25:1) as the eluent. After removing the solvents by a rotary
evaporator, the final product was obtained as yellow oil. The
yield was 51.6 % (0.32 g).
1H NMR (500 MHz, CDCl3, d): 8.53 (s, 3H, Ar H), 8.11–8.15 (m,
6H, Ar H), 7.93–7.95 (m, 8H, Ar H), 7.56–7.63 (m, 12H, Ar
H),3.98 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3, d): 166.53,
166.21, 139.54, 134.71, 131.51, 130.43, 128.20, 127.71, 117.12,
113.16, 52.03. IR (KBr): 3448 (NAH), 2951, 2844 (CAH), 1773
(C@O), 1716 (C@O), 1597, 1443, 1428 (Ar), 1235 (CAO), 730
cm21 (Ar). ESIMS (m=z (%)): 873 [M 1 Na]1.
Synthesis of G2-CH2OH
G2-CH2OH was synthesized by the same general procedure
described for the synthesis of G1-CH2OH. From G2-COOCH3
(0.41 g, 0.48 mmol), NaBH4 powder (0.55 g, 1.44 mmol) and
methanol (20 mL) were obtained 0.37 g (94.9%) as a gray-
ish solid.
1H NMR (500 MHz, CDCl3, d): 7.29–7.54 (m, 13H, Ar H), 5.16
(s, 2H, CH2), 3.70–4.21 (m, 4H, CH2), 1.60 (s, 6H, CH3), 1.24–
1.29 (m, 6H, CH3). 13C NMR (125 MHz, CDCl3, d): 193.15,
174.99, 174.31, 136.49, 128.65, 128.36, 128.25, 67.80, 62.92,
55.05, 48.53, 26.46, 22.14, 11.93. IR (KBr): 3421 (NAH), 2974,
2934 (CAH), 1731 (C@O), 1632 (C@O), 1488, 1458 (Ar),
1267, 1160 (CAO), 1012 cm21 (SAC@S). ESIMS (m=z (%)):
586 [M 1 Na]1.
1H NMR (500 MHz, DMSO-d6, d): 10.33 (s, 6H, CONH), 8.25
(s, 3H, Ar H), 8.00–8.01 (m, 6H, Ar H), 7.89–7.92 (m, 8H, Ar
H), 7.54–7.62 (m, 12H, Ar H), 4.54 (s, 2H, CH2).
13C NMR (125 MHz, DMSO-d6, d): 166.32, 144.07, 139.97,
135.85, 132.35, 129.19, 128.55, 115.14, 112.41, 63.90. IR
(KBr): 3453 (NAH), 2927, 2852 (CAH), 1625 (C@O), 1427
(Ar), 1195, 1140 (CAO), 842, 712 cm21 (Ar). ESIMS (m=z
(%)): 845 [M 1 Na]1.
Synthesis of G1-b-PNVCL
A Schlenk flask was charged with the [G-1]-based RAFT agent
(25 mg, 0.044 mmol), AIBN (3 mg, 0.018 mmol), and NVCL
(614 mg, 4.4 mmol); it was then degassed by three freeze–
evacuate–thaw cycles, back-filled with N2, and then placed in
an oil bath thermostated at 75 ꢀC to start the polymerization.
After 12 h, the reaction was aerated to quench, cooled to room
temperature, and then the mixture was precipitated into petro-
Synthesis of the [G-2]-based RAFT Agent
[G-2]-based RAFT agent was synthesized by the same general
procedure described for the synthesis of [G-1]-based RAFT
agent. From N,N-diethyl-S-(a,a0-dimethyl-a0-actetic acid)
dithiocarbamate (0.34 g, 1.41 mmol), G2-CH2OH (0.37 g,
0.45 mmol), DMAP (0.035 mg) and DCC (0.33 g, 1.55 mmol)
were obtained 0.21 g (45.0%) as yellow oil after purification
by silica gel column chromatography using petroleum ether-
ethyl acetate (v=v 5 35:1) as the eluent.
ꢀ
leum ether (b.p., 30–60 C) three times. The polymer collected
was dissolved in deionized water and then transferred to a di-
alysis bag (molecular weight cutoff [MWCO] 5 3500) and dia-
lyzed against deionized water for 6 days. The final product was
dried in a vacuum oven, yielding a light yellow-colored solid.
IR (KBr): 3449 (NAH), 2927, 2856 (CAH), 1632 (C@O),
1480, 1443 (Ar), 1263, 1151 (CAO), 1084 (SAC@S), 842,
716 cm21 (Ar).
(500 MHz, CDCl3, d): 8.09–8.28 (m, 9H, CH), 7.55–7.66 (m,
12H, CH), 5.16 (s, 2H, CH2), 3.71–4.27 (m, 4H, CH2), 1.61 (s,
6H, CH3), 1.26–1.30 (m, 6H, CH3). IR (KBr): 3422 (NAH),
2974, 2935 (CAH), 1730 (C@O), 1652 (C@O), 1632, 1487,
1461 (Ar), 1267, 1162 (CAO), 1074 (SAC@S), 832, 713
cm21 (Ar). ESIMS (m=z (%)): 1062 [M 1 Na]1.
Synthesis of G2-COOH
SOCl2 (1.1 g, 9.2 mmol) was added dropwise to a solution of
G1-COOH (3.6 g, 10 mmol) in 20 mL of NMP in ice-water
bath. After stirring in ice-water bath for 30 min, the mixture
was stirred for another 30 min at room temperature. Then,
3,5-diaminobenzoic acid (0.73 g, 4.8 mmol) was added to
the solution, and the reaction was carried out for 4 h at
Synthesis of G2-b-PNVCL
[G-2]-PNVCL was synthesized by the same general procedure
described for the synthesis of [G-1]-PNVCL agent. From
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[G-2]-based RAFT agent (46 mg, 0.044 mmol), AIBN (3 mg,
0.018 mmol) and NVCL (614 mg, 4.4 mmol) were obtained
as a light yellow-colored solid.
a 0.45-lm filter prior to use. Each reported measurement
was the average of three runs.
Drug Loading and In Vitro Release
IR (KBr): 3445 (NAH), 2928, 2855 (CAH), 1632 (C@O),
1479, 1443 (Ar), 1263, 1151 (CAO), 1083 (SAC@S), 842,
717 cm21 (Ar).
Gn-b-PNVCL copolymers (30 mg) and IMC (12 mg) were dis-
solved in 10 mL of DMF. The solution was poured into a di-
alysis bag (MWCO 5 3500) and dialyzed against 1000 mL of
distilled water for 24 h. The precipitate of unbound IMC was
removed by centrifugation at 4000 rpm for 30 min. The
micelles were obtained by vacuum drying. The addition of a
tenfold excess volume of acetone disrupted a weighed
amount of micelle. The amount of IMC entrapped into the
copolymer micelles was assayed with a TU-1901 UV–vis
spectrophotometer at 320 nm, using a standard calibration
curve experimentally obtained with IMC–acetone solutions.
The drug loading content (DLC) and entrapment efficiency
(EE) were calculated from the following equations50: DLC
(wt %) 5 (mass of IMC in micelles=mass of IMC 2 loaded
micelles) 3 100%; EE (%) 5 (mass of IMC in micelles=mass
of IMC initially loaded) 3 100%.
Micelle Formation
The micelles of the copolymers were prepared by a dialysis
method. Briefly, G1-b-PNVCL or G2-b-PNVCL was dissolved in
THF. The solution was put into a dialysis bag (MWCO 5
3500) and dialyzed against distilled water at room tempera-
ture for 24 h. After dialysis, the solution in the dialysis bag
was collected and vacuum dried for 72 h. In brief, 10 mg of
dried micelles was dispersed in 10 mL of distilled water. The
micellar solution was allowed for duration of 1 week before
further studies.
Transmission Electron Microscope Measurements
A drop of micellar solution (1 mg=mL) was placed on a FOR-
MVAR-coated copper grid and dried before measurement by
JEM-2100 transmission electron microscope (TEM) at an
acceleration voltage of 200 kV.
To study in vitro drug release, the dialysis bags were directly
placed in phosphate-buffered solutions (PBS) (pH 7.4) at 25
ꢀ
and 41 C, respectively. At predetermined time intervals, ali-
quots of 5 mL of buffered solution outside the dialysis bag
are removed for UV–vis analysis and replaced with the same
volume of a fresh buffer solution. The amount of IMC
released from micelles was measured by UV absorbance at
320 nm.
Fluorescence Measurements
Fluorescence measurements were conducted with a Hitich F-
4500 luminescence spectrophotometer using pyrene as a flu-
orescent probe as described previously.48,49 Aliquots of py-
rene solution (3.0 3 1025M in acetone, 50 lL) were added
to volumetric flasks. After complete acetone evaporation,
aqueous solutions of the copolymers at different concentra-
tions were added to the flasks to make the pyrene concen-
tration of 6 3 1027M. The solutions containing pyrene were
kept at room temperature for 24 h before measurements.
The emission spectra were recorded from 250 to 500 nm
with an excitation wave length of 390 nm.
RESULTS AND DISCUSSION
Synthesis of the Dendritic RAFT Agents
The synthesis procedures for the dendritic CTAs are shown
in Scheme 1. 3,5-Diaminobenzoic acid was reacted with ben-
zoyl chloride in NMP to afford the first-generation carboxylic
acid G1-COOH. The second-generation carboxylic acid G2-
COOH was synthesized via a two-step method consisting of
(1) activation of a G1-COOH by SOCl2, that is, generation of
an active amide and (2) condensation of this active amide
with 3,5-diaminobenzoic acid according to Ueda’s proto-
col.51,52 Gn-COOH were converted into methyl esters and
reduced to the corresponding benzylic alcohol Gn-CH2OH
using a sodium borohydride–THF–methanol system. This
two-step procedure allowed the selective reduction of esters
in the presence of amide functions.53 The dendritic CTAs
(Gn-CTA) were prepared by DCC=DMAP-mediated esterifica-
tion of Gn-CH2OH with N,N-diethyl-S-(a,a0-dimethyl-a00-actetic
acid)dithiocarbamate. We used 1H NMR and 13C NMR spec-
troscopy, FTIR spectroscopy, and ESI-MS to confirm the
structure of all new compounds.
Measurement of Transmittance
The LCST was determined by the cloud point measurement.
The copolymers were dissolved in deionized water at differ-
ent concentrations (0.1–4.0 mg=mL). Optical transmittance of
each copolymer solution was monitored as a function of tem-
perature at 500 nm with a UV–vis spectrometer (CARY UV-
50, VARIAN) equipped with a water-circulation heating stage.
The heating rate was 1 ꢀC=5 min. The LCST was defined as
the temperature corresponding to the initial break points in
the resulting transmittance versus temperature curve.
Dynamic Light Scattering
Dynamic light scattering (DLS) measurements were per-
formed with a Zetasizer ZEN 3600 instrument (Malvern,
These CTAs possess a single dithiocarbamate functional group
at the focal point of the dendron, allowing a straight forward
RAFT approach to linear–dendritic block copolymers.
ꢀ
United Kingdom) operating at 20–50 C using a light scatter-
ing apparatus equipped with a He–Ne laser. The scattering
angle was kept at 173ꢀ (backscattering) and the wave length
in the vacuum was set as 633 nm during the whole experi-
ment. Malvern DTS 6.20 software was used to analyze the
data. The micellar solutions (1.0 mg=mL) were filtered with
Synthesis of Linear–Dendritic Copolymer
We recently reported the synthesis of PNVCL with a con-
trolled molecular weight by RAFT polymerization with AIBN
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as an initiator and dithiocarbamate or trithiocarbonate as a
RAFT agent.37 Here, the dendron Gn-CTA was used as RAFT
agent to polymerize NVCL, yielding polyamide–PNVCL den-
dritic–linear block copolymers. The products were character-
1
ized by FTIR, H NMR spectra (Figs. 1 and 2), and GPC. The
GPC traces (Fig. 3) of the copolymers are unimodal and sym-
metric, and show that no detectable shoulder peak at high
MW position attributed to linear–linear coupling polymer
was observed. The polydispersities of G1-b-PNVCL and G2-b-
PNVCL were determined by GPC=MALLS to be 1.21 and 1.30,
respectively. It can be calculated from the GPC results that
G1-b-PNVCL and G2-b-PNVCL have DPs of 46 and 55, respec-
tively, which were in fairly good agreement with the theoreti-
cal DP (Table 1).
Formation of Micelles from Amphiphilic
Linear–Dendritic Hybrid Block Copolymers
The amphiphilic nature of block copolymers, consisting of
hydrophilic PNVCL and hydrophobic aromatic polyamide
dendron blocks, provides an opportunity to form micelles in
water. The characteristics of block copolymer micelles in an
aqueous phase were investigated by fluorescence techniques
using pyrene as a probe. Figure 4(a,b) shows the emission
spectra of pyrene in the G1-b-PNVCL and G2-b-PNVCL solu-
tions at various concentrations, respectively. Red shift of py-
rene in the emission spectra was observed with increasing
concentration of the copolymers. Figure 5(a,b) shows the in-
tensity ratio of I383=I373 versus the logarithm of concentra-
tions of G1-b-PNVCL and G2-b-PNVCL in the pyrene emission
SCHEME 1 Synthesis of well-defined block copolymers con-
taining polyamide dendrons and PNVCL chains (Gn-b-PNVCL).
FIGURE 1 1H NMR spectra of G1-b-PNVCL in CDCl3 (a), DMSO-d6 (b), and D2O (c).
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FIGURE 2 1H NMR spectra of G2-b-PNVCL in CDCl3 (a), DMSO-d6 (b), and D2O (c).
spectra. An abrupt increase in the total fluorescent intensity
was observed with increasing copolymer concentrations,
indicating the formation of micelles and the transfer of py-
rene into the hydrophobic core of micelles. The concentra-
tion at the abrupt increase in the ratio of I383=I373 was
defined as the critical micelle concentration (CMC). The CMC
values of G1-b-PNVCL and G2-b-PNVCL were 0.1 and 0.017
mg=mL, respectively. The CMC of G1-b-PNVCL is much higher
owing to the small portion of the hydrophobic block,
whereas that of G2-b-PNVCL is comparable with those
reported for some other amphiphilic linear–dendritic hybrid
block copolymers and linear copolymers. As the hydrophobic
dendritic composition became higher, a lower CMC value was
observed.
hydrophobic inner cores and hydrophilic outer shells. Similar
trends of 1H NMR spectra are consistent with amphiphilic
linear block copolymer systems.54,55
Morphology of the polymeric micelles was investigated by
transmission electron microscope (TEM). As shown in Figure
6(a), G1-b-PNVCL forms spherical micelles and the total size
The formation of micelles from G1-b-PNVCL and G2-b-PNVCL
1
in aqueous solution was verified by H NMR spectra. Figures
1(a,b) and 2(a,b) show that the complete structural resolu-
tion of each block was observed in CDCl3 and DMSO-d6,
respectively, two nonselective solvents for G1 or G2 and
PNVCL blocks. But the 1H NMR spectra in D2O [Figs. 1(c)
and 2(c)] showed that the characteristic signals of G1 or G2
block at 7.1–8.1 ppm disappeared owing to the limited mo-
bility of G1 or G2 block in the core of the nanoparticles. The
results revealed that Gn-b-PNVCL diblock copolymers may
associate to form the core–shell micellar structure with
FIGURE 3 GPC traces of Gn-b-PNVCL.
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TABLE 1 Characteristics of Gn-b-PNVCL
Conv.a Mnthb Mn
(Fig. 7). DLS of G1-b-PNVCL micelles confirmed micellar diam-
eters ranging from 70 to 170 nm, having a broad size distri-
bution. The diameter of G2-b-PNVCL micelles ranged from 50
to 80 nm. The distribution of the micelles was narrow. These
results are consistent with the TEM images, considering that
the DLS result is for micelles in an aqueous solution. G1-b-
PNVCL formed larger aggregates than G2-b-PNVCL in water.
The reason may arise from the differences in hydrophilicity
between G1-b-PNVCL and G2-b-PNVCL. Compared with G2-b-
PNVCL, G1-b-PNVCL possesses more hydrophilic PNVCL
chains. The more water-soluble PNVCL segments tend to bind
more water molecules and swell the aggregated copolymer
chains, resulting in the formation of the larger aggregates.
c
LCST
Copolymers CTA
(%)
(g=mol) (g=mol) PDIc (ꢀC)d
G1-b-PNVC G1-CTA 45
6,818
8,684
6,904
8,990
1.21 44 39
LG2-b-PNVCL G2-CTA 55
1.30 39 34.5
a
Monomer conversion as determined by gravimetric method.
b
The theoretic molecular weight was calculated by the formula: Mnth 5
100 3 139.2 3 conv. 1 molecular weight of Gn-CTA.
c
Mn and PDI were determined by GPC=MALLS.
LCSTs of Gn-b-PNVCL in deionized water and in PBS, respectively, at
d
a concentration of 1 mg=mL were measured by turbidity measurements
using UV–vis spectroscopy.
Thermoresponsive Properties
The thermoresponsive properties of these micellar structures
were investigated by turbidity measurements. Figure
shows the transmittances of aqueous solutions of linear–den-
dritic copolymers as a function of temperature with various
concentrations. The linear–dendritic copolymers showed rel-
atively sharp transitions during the heating processes, and
of the observed micelles is not uniform with diameters rang-
ing from 60 to 150 nm. The micelles are interconnected or
partially overlapped with one another. In Figure 6(b), self-
assembled G2-b-PNVCL micelles dispersed as individual nano-
particles with a regularly spherical shape with a diameter of
40–60 nm. The size and size distribution of the self-assembled
micelles were further examined by DLS at room temperature
8
ꢀ
their LCSTs (ranging from 46 to 41 C for G1-b-PNVCL, from
ꢀ
42 to 36 C for G2-b-PNVCL) decreased with increasing con-
centration from 0.1 to 4.0 mg=mL. Compared to linear
FIGURE 5 Plot of I383=I373 versus logarithm of concentration of
G1-b-PNVCL (a) and G2-b-PNVCL (b).
FIGURE 4 Fluorescence spectra of pyrene in water at different
concentrations of G1-b-PNVCL (a) and G2-b-PNVCL (b).
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FIGURE 6 TEM images of blank G1-b-PNVCL micelles (a), blank G2-b-PNVCL micelles (b), IMC-loaded G2-b-PNVCL micelles (c),
and G2-b-PNVCL micelles after drug release at 41 ꢀC for 60 h (d).
PNVCL with similar MW,37 G1-b-PNVCL shows higher LCSTs
at all concentration tested. Presumably, the G1 dendron pos-
sessed amide moieties that could interact with adjacent
water molecules through hydrogen bonds, which enhanced
the hydrophilicity of G1-b-PNVCL and conduced to a higher
LCST. The validation of this hypothesis is currently ongoing
in our laboratories. The LCSTs of G2-b-PNVCL are lower than
ones of G1-b-PNVCL at all concentrations tested, demonstrat-
ing the dependence of LCSTs on the generation of linear–
dendritic copolymers. It is well known that the LCST of a
thermoresponsive polymer depends mostly on the hydropho-
bicity–hydrophilicity balance. In general, the more hydropho-
bic polymers show lower LCSTs than the more hydrophilic
ones. For G1-b-PNVCL and G2-b-PNVCL, the G1 and G2
blocks afforded them with different hydrophilic and hydro-
phobic compositions. G2 dendron possessed more hydropho-
bic phenyl groups, which would enhance the hydrophobicity
of G2-b-PNVCL and thus resulted in a lower LCST. Although
G2 dendron possessed more amide moieties, they were bur-
ied inside the hydrophobic core of the micelles, which could
not interact with adjacent water molecules through hydrogen
bonds. The CMC values of the linear–dendritic copolymers
measured above also indicate that G2-b-PNVCL is more
hydrophobic than G1-b-PNVCL.
(Dh) upon heating from 20 to 50 C in intervals of 1 C. Fig-
ure 9 shows that the hydrodynamic diameter (Dh) of the
copolymers is apparently influenced by the temperature. At
temperature lower than the LCSTs, the micelles were stable
having Dh of approximately 55–130 nm. However, when
heated to 44 and 39 ꢀC, the Dh of the micelles increased to
500 and 1200 nm, respectively, because the shell of the
micelles became hydrophobic at temperatures above the
LCST, leading to intermicellar hydrophobic association. G1-b-
PNVCL requires higher temperature to induce aggregation
than G2-b-PNVCL does. The aggregation process was com-
pletely reversible, and the aggregates redispersed, and the
size reduced to the original level upon cooling. These are in
good agreement with the results from turbidimetry
measurement.
ꢀ
ꢀ
PBSs are known to affect the phase behavior of PNVCL
aqueous solution.29 To investigate the effect of PBS on the
LCSTs of Gn-b-PNVCL, the phase transitions of the copoly-
mers in PBS were examined with UV=vis spectroscopy. The
LCST of G1-b-PNVCL and G2-b-PNVCL in PBS at a concen-
ꢀ
tration of 1 mg=mL was found to be at 39 and 34.5 C (Ta-
ble 1), respectively, which is lower than that in deionized
water at the same concentration (44 and 39 ꢀC for G1-b-
PNVCL and G2-b-PNVCL, respectively). This phenomenon is
consistent with PNVCL-containing amphiphilic linear block
copolymers.29
The thermoresponsive behavior of the copolymers was also
analyzed by DLS to determine the hydrodynamic diameters
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FIGURE 7 Size distributions of G1-b-PNVCL (a) and G2-b-
FIGURE 8 Transmittance measurements as a function of tem-
perature for different concentrations of G1-b-PNVCL (a) and
G2-b-PNVCL (b).
PNVCL (b) micelles at room temperature.
Controlled Drug Release
It is well known that hydrophobic drugs could be loaded in
the micelles owing to the hydrophobic core of the micelle.
IMC is an anti-inflammatory drug having poor solubility in
water. In view of the thermoresponsive property of Gn-b-
PNVCL micelles, we utilized self-assembly of Gn-b-PNVCL
and IMC to prepare IMC-loaded micelles in distilled water by
a dialysis technique, and evaluated their possible application
for drug delivery. The amount of drug incorporated into the
micelles was measured by UV spectrometer. The results
showed that IMC was successfully incorporated into micelles
of G1-b-PNVCL and G2-b-PNVCL, and the DLC and EE were
about 20.25 and 73.64% for G1-b-PNVCL, 25.76 and 85.19%
for G2-b-PNVCL, respectively. The DLC and EE of G2-b-PNVCL
are higher than those of G1-b-PNVCL. This can be ascribed
to the following reason: with the increase in generations of
micelles, there was a significant decrease in CMCs; also,
there is an increase in hydrophobic phenyl groups in the
micelles for hydrophobic interactions, leading to increase in
drug loading.
FIGURE 9 Plots of hydrodynamic diameters (Dh) of G1-b-
PNVCL (a) and G2-b-PNVCL (b) micelles in aqueous solution as
a function of temperature from DLS measurements.
As shown in Figure 6(c), a TEM image of IMC-loaded
micelles made from G2-b-PNVCL reveals that the addition of
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CONCLUSIONS
We have successfully synthesized the first- and second-gen-
eration well-defined thermoresponsive amphiphilic linear–
dendritic diblock copolymers (Gn-b-PNVCL) with linear
PNVCL as hydrophilic block and dendritic aromatic polyam-
ide as hydrophobic block by RAFT polymerization. The syn-
thesized copolymers exhibit reversible and fast tranꢀsitions in
aqueous solution with LCSTs in the range of 36–46 C, which
depend on the generation of the dendrons and the concen-
trations of the copolymer solutions. The block copolymers
formed nanospherical micelles in the aqueous phase owing
to their amphiphilic characteristics. The CMC of G1-b-PNVCL
is 0.1 mg=mL, whereas G2-b-PNVCL has a CMC of 0.017
mg=mL. Hydrophobic drugs can been capsulated in the
micelles. Drug EE and DLC increased with increasing genera-
tion of the linear–dendritic copolymers. In vitro release tests
showed a desired thermoresponsive drug release behavior.
FIGURE 10 In vitro release profile of IMC from G1-b-PNVCL
and G2-b-PNVCL micelles at 25 and 41 ꢀC, respectively.
IMC to the micelle core initiates significant aggregation of
the micelles. Similar phenomenon has been observed for
paclitaxel-loaded micelles of linear–dendritic hybrids com-
posed of PCL arms on a bis-MPA dendron and a linear com-
ponent of PEG.6
ACKNOWLEDGMENTS
The authors gratefully acknowledge support for this work of
the National Natural Science Foundation of China (20864003
and 21264017), Program for Changjiang Scholars and Innova-
tive Research Team in University, PCSIRT, and the Natural Sci-
ence Foundation of Yunnan Province (2009ZC064M), People’s
Republic of China.
The applicability of G1-b-PNVCL and G2-b-PNVCL micelles in
controlled drug release was examined in PBS (pH 5 7.4).
The drug release profiles from micelles are shown in Figure
10. It was clearly observed that the initial burst was fol-
lowed by a controlled drug release from the micelles. The
initial burst release could be ascribed to IMC molecules
located within the shell or at the interface between the mi-
celle core and the shell. As shown in Figure 8, the rate of
drug release was found to be faster above the LCST than
that below the LCST of Gn-b-PNVCL. This can be explained
by the core–shell micellar structure and the thermorespon-
sive properties of Gn-b-PNVCL. When the temperature was
below the LCST, the core–shell structure was stable and IMC
release depended on its slow diffusion. However, when the
temperature was raised above the LCST, PNVCL segments in
the shells of the micelles collapsed. The collapsed shell
induced the deformation of the core–shell structure, and
thus accelerating IMC release. To confirm the deformation of
micelles, the TEM measurement of G2-b-PNVCL micelles after
drug release at 41 ꢀC for 60 h was preformed. As shown in
Figure 6(d), incomplete spherical micelles coexist with
vesicles of irregular shape, having a size in the range of
120–260 nm, were observed. This result demonstrates that
the morphology and size of G2-b-PNVCL micelles changed af-
ter drug release.
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