Synthesis and properties of multicleavable amphiphilic dendritic comblike and toothbrushlike copolymers comprising alternating PEG and PCL grafts

Article abstract of DOI:10.1021/ma3025283

Facile construction of novel functional dendritic copolymers by combination of self-condensing vinyl polymerization, sequence-controlled copolymerization and RAFT process was presented. RAFT copolymerization of a disulfide-linked polymerizable RAFT agent and equimolar feed ratio of styrenic and maleimidic macromonomers afforded multicleavable A_mB_n dendritic comblike copolymers with alternating PEG (A) and PCL (B) grafts, and a subsequent chain extension polymerization of styrene, tert-butyl acrylate, methyl methacrylate, and N-isopropylacrylamide gave A_mB _nC_o dendritic toothbrushlike copolymers. (PEG) _m(PCL)_n copolymers obtained were of adjustable molecular weight, relatively low polydispersity (PDI = 1.10-1.32), variable CTA functionality (f_CTA = 4.3-7.5), and similar segment numbers of PEG and PCL grafts, evident from ¹H NMR and GPC-MALLS analyses. Their branched architecture was confirmed by (a) reduction-triggered degradation, (b) decreased intrinsic viscosities and Mark-Houwink-Sakurada exponent than their linear analogue, and (c) lowered glass transition and melting temperatures and broadened melting range as compared with normal A _mB_n comblike copolymer. In vitro drug release results revealed that the drug release kinetics of the disulfide-linked A _mB_n copolymer aggregates was significantly affected by macromolecular architecture, end group and reductive stimulus. These stimuli-responsive and biodegradable dendritic copolymer aggregates had a great potential as controlled delivery vehicles.

Full text of DOI:10.1021/ma3025283

Article
pubs.acs.org/Macromolecules
Synthesis and Properties of Multicleavable Amphiphilic Dendritic
Comblike and Toothbrushlike Copolymers Comprising Alternating
PEG and PCL Grafts
Meijing Zhang,† Huanhuan Liu,† Wei Shao, Ke Miao, and Youliang Zhao*
Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering,
College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China
S
* Supporting Information
ABSTRACT: Facile construction of novel functional den-
dritic copolymers by combination of self-condensing vinyl
polymerization, sequence-controlled copolymerization and
RAFT process was presented. RAFT copolymerization of a
disulfide-linked polymerizable RAFT agent and equimolar feed
ratio of styrenic and maleimidic macromonomers afforded
multicleavable AmBn dendritic comblike copolymers with
alternating PEG (A) and PCL (B) grafts, and a subsequent
chain extension polymerization of styrene, tert-butyl acrylate, methyl methacrylate, and N-isopropylacrylamide gave AmBnCo
dendritic toothbrushlike copolymers. (PEG)m(PCL)n copolymers obtained were of adjustable molecular weight, relatively low
polydispersity (PDI = 1.10−1.32), variable CTA functionality (fCTA = 4.3−7.5), and similar segment numbers of PEG and PCL
grafts, evident from 1H NMR and GPC-MALLS analyses. Their branched architecture was confirmed by (a) reduction-triggered
degradation, (b) decreased intrinsic viscosities and Mark−Houwink−Sakurada exponent than their “linear” analogue, and (c)
lowered glass transition and melting temperatures and broadened melting range as compared with normal AmBn comblike
copolymer. In vitro drug release results revealed that the drug release kinetics of the disulfide-linked AmBn copolymer aggregates
was significantly affected by macromolecular architecture, end group and reductive stimulus. These stimuli-responsive and
biodegradable dendritic copolymer aggregates had a great potential as controlled delivery vehicles.
reaction to synthesize hyperbranched macroinitiators, dendritic
polymer brushes, and star-shaped polymers.43 With the
development in advanced polymer synthesis, it is extremely
urgent to further apply hyperbranched copolymers as versatile
scaffolds to construct novel dendritic and starlike copolymers
via postmodification and postpolymerization in addition to
pursuing for ideal approaches to rapid synthesis of the target
copolymers.
Meanwhile, much emphasis has been paid to sequence-
regulated polymerization since the introduction of regular
microstructures involving alternating AB, AAB, and ABA
sequences into well-defined copolymers usually endows
functional materials with new features such as novel self-
assembled nanoobjects, unique degradation and crystallization
behaviors and tunable amphiphilic properties.44−67 To date, a
series of copolymers comprising precise microstructures have
been successfully achieved by chain growth polymerization.
Among them, maleimidic and styrenic monomers are generally
liable to alternating copolymerization, and thus numerous
alternating and sequence-controlled copolymers,52−57 copoly-
mer brushes,58−62 and dendritic-linear block copolymers with
alternating AB sequences63−65 have been precisely prepared by
INTRODUCTION
■
Facile construction of complex macromolecular architectures
such as dendritic and hyperbranched copolymers,1−10 star
polymers11−21 and polymer brushes22−30 have attracted much
attention due to their unique physicochemical properties and
multipurpose applications originating from the branched
topologies. As a subclass of dendritic polymers, segmented
hyperbranched copolymers comprising long linear branched
chains are of increasing interest due to their wide chemical
composition, adjustable degree of branching and chain length,
and ability for versatile postfunctionalization. Generally speak-
ing, self-condensing vinyl polymerization (SCVP)31 via
controlled radical processes such as atom transfer radical
polymerization (ATRP)32−36 and reversible addition−fragmen-
tation chain transfer (RAFT) polymerization37−43 can be
efficiently used to achieve hyperbranched architectures with
variable molecular parameters. RAFT SCVP was previously
used by us to achieve hyperbranched copolymers with variable
chain transfer agent (CTA) functionality and relatively low
polydispersity, and a variety of multiarm and miktoarm star
polymers with a branched core were further generated by
subsequent chain extension polymerization and/or quaterniza-
tion reaction between branched scaffolds and bromide-
functionalized polymers.41,42 More recently, Gao and co-
workers ingeniously combined RAFT SCVP, Menschutkin
reaction and Cu(I) catalyzed azide−alkyne cycloaddition
Received: December 9, 2012
Revised: January 31, 2013
Published: February 11, 2013
© 2013 American Chemical Society
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Scheme 1. Synthetic Routes to Multicleavable Amphiphilic AmBn-Type Dendritic Comblike Copolymers and AmBnCo-Type
Dendritic Toothbrushlike Copolymers
radical copolymerization. Chen and co-workers previously
reported both (a) atom transfer radical copolymerization of
maleimidic inimer with St and (b) controlled radical
copolymerization of bismaleimide with excess St could generate
multiarm PSt stars via one-pot process, revealing the great
potential of alternating copolymerization in novel architecture
construction.66,67 We used RAFT copolymerization of vinyl-
benzyl-terminated PEG (St-PEG) and N-(2-hydroxyethyl)
maleimide (HEMI) to synthesize well-defined symmetric
disulfide-linked A2mB2n-type comblike copolymers62 and
A2mB2nC2-type starlike terpolymers20 with alternating poly-
(ethylene glycol) (PEG, A) and poly(ε-caprolactone) (PCL, B)
pendent chains. These copolymers could be efficiently
converted into degraded thiol-terminated comblike and starlike
copolymers upon reduction, and followed by oxidation to form
disulfide-linked high-molecular-weight copolymer brushes and
comblike-linear multiblock copolymers; such redox cycles could
be efficiently repeated.20,62 To our surprise, the examples of
hyperbranched and dendritic copolymers with regular sequen-
ces are very scarce thus far.68,69 Only a few hyperbranched
alternating block copolymers were prepared by Perrier and co-
workers via thiol−yne “click” chemistry, in which RAFT
synthesized clickable diblock copolymers were used as
precursors to linear branches.68,69 At present, accelerated
construction of new functional copolymers via sequence-
controlled polymerization is of great importance since it not
only introduces intriguing phase morphologies and multiple
stimuli-responsive properties but has promising applications in
smart biomaterials and nanotechnologies.
Herein, we report on facile synthesis of novel multicleavable
amphiphilic dendritic comblike and toothbrushlike copolymers
with alternating PEG and PCL grafts by combination of self-
condensing vinyl polymerization, sequence-controlled copoly-
merization and RAFT process (Scheme 1). RAFT copoly-
merization of styrenic and maleimidic macromonomers
mediated by 2-((2-(acryloyloxy)ethyl)disulfanyl)ethyl 4-
cyano-4-(phenylcarbonothioylthio)pentanoate (ACP) afforded
AmBn-type dendritic comblike copolymers with alternating PEG
(A) and PCL (B) grafts in each branch and cleavable disulfide
moiety in each branching point, and AmBnCo-type dendritic
toothbrushlike copolymers were further generated by AmBn
copolymer mediated chain extension polymerization. The
resultant dendritic copolymers were characterized by 1H
NMR, GPC-MALLS, DSC and viscosity measurement. Mean-
while, stimuli-triggered drug loading and release from the self-
assembled AmBn copolymer aggregates were preliminarily
investigated. To the best of our knowledge, this is the first
example on facile synthesis of dendritic comblike copolymers
with high-density alternating grafts on the basis of sequence-
controlled copolymerization. The methodology developed in
this study has a great potential in accelerated construction of
versatile thiol-responsive AmBn and AmBnCo (m ≈ n) dendritic
copolymers with rich chemical composition, tunable dithio-
benzoate and disulfide functionalities, and variable number and
chain length of C segments. In addition, the presence of
terminal hydroxyl functionalities in PCL segments allows for
chain extension polymerization and versatile postmodification
to introduce different varieties of polymer segments and
functionalities.
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Table 1. Synthesis of AmBn Dendritic Comblike Copolymers (DC1-DC8) and Normal Comblike Copolymer (C1) by RAFT
a
Copolymerization of St-PEG and MI-PCL Mediated by ACP (Runs 1-8) and CPDB (Run 9)
b
b
c
c
d
e
f
f
run
sample
DPPCL
x
CCTA (%)
C%
Mn,LS
PDI
[η]w (mL/g)
f CTA
m
n
1
2
3
4
5
6
7
8
9
DC1
DC2
DC3
DC4
DC5
DC6
DC7
DC8
C1
8.2
8.2
5
93.3
95.8
99.5
54.6
25.9
19.6
29.4
51.1
99.7
56.8
35.2
50.8
39.2
35.1
28.5
35.2
30.3
53.9
46 600
78 300
136 000
62 000
148 600
312 000
52 100
89 500
22 500
1.32
1.10
1.21
1.20
1.19
1.20
1.23
1.28
1.22
13.5
15.1
17.6
9.2
7.5
5.4
7.0
6.8
4.3
4.3
6.8
5.0
22.5
39.2
68.6
24.5
59.8
21.8
38.6
67.2
23.8
61.1
20
20
5
8.2
12.6
12.6
12.6
43.2
43.2
8.2
10
20
1
13.6
15.2
20.1
26.5
11.6
125
126
8.5
14.9
11.5
8.2
14.6
11.2
5
20
0.96
a
Polymerization conditions: [St-PEG]0:[MI-PCL]0:[CTA]0:[AIBN]0 = x:x:1:0.2, [MI-PCL]0 = 0.20 (runs 1−3 and 9) or 0.05 mol/L (runs 4−8), in
b
dioxane at 80 °C for 48 (runs 3 and 9) or 24 h (other runs). Total monomer conversion (C) and ACP conversion (CCTA) determined by
combination of gravimetry and H NMR analysis. Number-average molecular weight and polydispersity determined by GPC−MALLS. Weight-
average intrinsic viscosity. Number-average CTA functionality per copolymer. Number-average segment numbers of PEG (m) and PCL (n) grafts
c
d
1
e
f
per copolymer.
Synthesis of MI-PCL Macromonomers. In a typical reaction
(run 1 of Table S1, Supporting Information), to a Schlenk tube were
added HTD (1.05 g, 5.0 mmol), CL (11.4 g, 100 mmol), Sn(Oct)2
(0.203 g, 0.50 mmol), and 22 mL of dry toluene under nitrogen. The
EXPERIMENTAL SECTION
■
Materials. All solvents, monomers, and other chemicals were
purchased from Sigma-Aldrich unless otherwise stated. ε-Caprolactone
(CL, 99%) and bis(2-hydroxyethyl) disulfide (Alfa Aesar, 90%) were
contents were stirred for 5 min and subjected to three freeze-vacuum-
distilled from calcium hydride under reduced pressure. Styrene (St,
thaw cycles, and then the tube was immersed into an oil bath at 110
99%), 4-vinylbenzyl chloride (VBC, 90%), methyl methacrylate
°C to perform polymerization. After 20 h, the polymerization was
(MMA, 99%) and tert-butyl acrylate (tBA, 98%) were passed through
quenched by putting the tube into an ice−water bath. About 0.1 mL of
a basic alumina column to remove the inhibitor before use. N-
Isopropylacrylamide (NIPAM, 97%) was recrystallized twice from
1
polymerization solution was drawn to check H NMR spectroscopy,
1
and monomer conversion was determined to be 79.2% by H NMR
mixtures of hexane and toluene. 2,2′-Azobis(isobutyronitrile) (AIBN)
analysis. The polymerization solution was concentrated and
was recrystallized twice from ethanol. Monomethoxy poly(ethylene
precipitated into a large amount of cold hexane. The isolated PCL
glycol) (MPEG, Mn = 750, Fluka) was dried by azeotropic distillation
was dissolved in 100 mL of anisole, and the solution was heated at 120
in the presence of toluene. Vinylbenzyl-terminated PEG (St-PEG),59
°C for 16 h. After concentration and precipitation, 9.2 g of MI-PCL
4-(2-hydroxyethyl)-10-oxa-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-
was obtained. The apparent molecular weight and polydispersity
dione (HTD),70 2-(2-cyanopropyl) dithiobenzoate (CPDB),37 4-
estimated by GPC were Mn,GPC = 1990 and PDI = 1.10. The number-
cyanopentanoic acid dithiobenzoate (4-CPDB),71 and 2-((2-
average molecular weight determined by 1H NMR was Mn,NMR = 1080,
hydroxyethyl)disulfanyl)ethyl 4-cyano-4-(phenylcarbonothioylthio)-
which was close to theoretical value (Mn,th = 1040). Other MI-PCL
pentanoate72 were synthesized and purified according to literature
samples were synthesized and purified according to similar procedures.
procedures. N,N′-Dicyclohexylcarbodiimide (DCC) and 4-dimethyla-
1H NMR (CDCl3): δ 6.76 (CHCH), 4.24 (NCH2CH2O), 4.06
minopyridine (DMAP) were purchased from Sinopharm Chemical
Reagent Co., Ltd. and used as received. D,L-Dithiothreitol (DTT, 99%,
Merck) and doxorubicin hydrochloride (>99%, Zhejiang Hisun
Pharmaceutical Co, Ltd.) were used as received. Dichloromethane
(DCM) and dioxane were dried and distilled over CaH2.
Tetrahydrofuran (THF) and toluene were distilled over sodium and
(CH2O of PCL), 3.80 (CH2N), 3.66 (CH2OH), 2.31 (CH2CO of
PCL), 1.65 and 1.40 (CH2CH2CH2CH2O of PCL). 13C NMR
(CDCl3): δ 173.40 (CO), 170.28 (CO of MI ring), 134.12
(CHCH), 63.98 (CH2O of CL unit), 62.22 (CH2OH), 61.17
(NCH2CH2O), 36.77 (NCH2), 33.95 (CH2CO of CL unit), 33.62
(CH2CO of terminal CL unit), 32.15 (CH2CH2OH), 28.16
(CH2CH2O of CL unit), 25.36 (CH2CH2CH2O of CL unit), 25.18
(CH2CH2CH2CH2OH), 24.55 (CH2CH2CH2OH), 24.41
(CH2CH2CO of CL unit). FT-IR (KBr): 3437, 2945, 2864, 1724,
1471, 1420, 1398, 1369, 1296, 1247, 1195, 1106, 1047, 963, 934, 839,
732, 697 cm−1.
benzophenone and stored under nitrogen.
Synthesis of 2-((2-(Acryloyloxy)ethyl)disulfanyl)ethyl 4-
Cyano-4-(Phenylcarbonothioylthio) pentanoate (ACP). To a
250 mL of round flask were added 2-((2-hydroxyethyl)disulfanyl)ethyl
4-cyano-4-(phenylcarbonothioylthio)pentanoate (2.49 g, 6.0 mmol),
acrylic acid (0.50 g, 6.9 mmol), DMAP (0.12 g, 1.0 mmol) and 100
mL of dry DCM under nitrogen, and followed by slow addition of a 30
mL of DCM solution with 1.55 g (7.5 mmol) of DCC to perform the
esterification. The mixture was further stirred at ambient temperature
overnight. The crude product was filtered, concentrated and purified
by flash column chromatography eluting with hexane/DCM (2:1), and
2.26 g (80.2% yield) of ACP was obtained as a red oil.
Synthesis of AmBn Dendritic Comblike Copolymers by RAFT
Self-Condensing Vinyl Copolymerization. In a typical experiment
(run 2 of Table 1), ACP (94.0 mg, 0.20 mmol), St-PEG (3.48 g, 4.0
mmol), MI-PCL (4.32 g, 4.0 mmol), and AIBN (6.6 mg, 0.04 mmol)
were added to a Schlenk tube, and dry dioxane was added until the
total volume was 20.0 mL. The contents were degassed with bubbled
nitrogen for 30 min, and then the polymerization was performed at 80
°C for 24 h. The polymerization solution was precipitated into diethyl
ether thrice, and 2.84 g (35.2% total monomer conversion) of
(PEG)m(PCL)n copolymer (denoted as DC2) was obtained after
vacuum drying. The conversion of ACP (CCTA) was determined to be
1H NMR (300 MHz, CDCl3): δ 7.92 (d, J 7.5, PhH, 2H), 7.56 (t, J
6.9, PhH, 1H), 7.39 (t, J 7.5, PhH, 2H), 6.46, 6.14, and 5.87 (m, CH
CH2, 3H), 4.41 and 4.38 (m, CH2O, 4H), 2.96 (dd, J 6.9, CH2S, 4H),
2.70, 2.62, and 2.45 (m, CH2CH2CO, 4H), 1.93 (s, CH3, 3H). 13C
NMR (75 MHz, CDCl3): δ 222.1 (CS), 171.1, 165.7 (CO),
144.3, 133.0, 131.3, 128.5, 127.9, 126.5 (PhC and CHCH2), 118.3
(CN), 62.6, 62.2 (CH2O), 45.6 (CH3CCN), 37.0, 36.9 (CH2S), 33.1,
29.6 (CH2), 24.0 (CH3). FT-IR (KBr): 3262, 3060, 2934, 2853, 2231,
1732, 1649, 1635, 1618, 1590, 1543, 1445, 1407, 1296, 1268, 1181,
1109, 1079, 1049, 983, 902, 868, 810, 763, 688, 650, 617 cm−1. Anal.
Calcd for C20H23NO4S4: C, 51.15; H, 4.94; N, 2.98; S, 27.31. Found:
C, 51.32; H, 4.96; N, 2.95; S, 27.09.
1
95.8% by combination of gravimetry and H NMR analysis. Number-
average molecular weight and polydispersity determined by GPC-
MALLS were Mn,LS = 78300 and PDI = 1.10. Based on GPC-MALLS
and NMR analyses, its number-average CTA functionality (f CTA) was
determined to be 5.4, and number-average segment number of each
graft was calculated to be mPEG = 39.2, and nPCL = 38.6. Other
dendritic comblike copolymers were synthesized and isolated
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Table 2. Synthesis of AmBnCo (A = PEG, B = PCL, C = PM) Dendritic Toothbrushlike Copolymers by RAFT Polymerization
a
Mediated by DC2 (Mn,LS = 78300, fCTA = 5.4)
b
c
d
d
e
f
g
run
M
DP0
C %
Mn,th
Mn,LS
PDI
Mn,NMR
dn/dc
[η]w (mL/g)
1
2
3
4
St
200
150
150
150
19.9
46.8
38.6
47.2
100 700
126 900
109 600
121 500
105 100
125 700
109 300
121 400
1.06
1.09
1.11
1.13
103 200
128 000
107 800
122 600
0.104
19.4
25.1
20.2
23.9
tBA
0.0758
0.0857
0.0886
MMA
NIPAM
a
Polymerization conditions: [M]0:(f CTA[macro CTA]0):[AIBN]0 = DP0:1:0.2, [M]0 = 1.5 mol/L, in toluene (runs 1−3) or dioxane (run 4) at 80 °C
b
c
for 12 h. Monomer conversion determined by gravimetry. Theoretical molecular weight, Mn,th = 78300 + f CTA × DP0 × conversion × MWM, in
d
which MWM was molecular weight of vinyl monomers. Number-average molecular weight and polydispersity determined by GPC-MALLS.
e
f
g
1
Number-average molecular weight determined by H NMR. Determined by refractive index detector. Weight-average intrinsic viscosity.
according to a similar approach. Besides, a normal AmBn comblike
copolymer (denoted as C1) was also prepared by RAFT
copolymerization of St-PEG and MI-PCL mediated by CPDB.
(PEG)m(PCL)n Dendritic Comblike Copolymer. 1H NMR (CDCl3):
δ 7.98 (PhH of terminal PhC(S)S), 6.2−7.8 (ArH of St-PEG unit
and PhH of terminal PhC(S)S), 4.8−5.1 (CHS), 4.50 and 4.24
(CH2O), 4.06 (CH2O of PCL), 3.87 (CH of MI-PCL unit), 3.65
(CH2CH2O of PEG), 3.56 (CH2N of MI-PCL unit), 3.38 (CH3O of
PEG), 0.8−3.0 (CH, CH2 and CH3 originating from RAFT agent,
maleimidic and styrenic units). FT-IR (KBr): 3464, 2939, 2867, 1728,
1702, 1638, 1466, 1398, 1352, 1296, 1246, 1193, 1105, 1041, 953, 849,
733, 669 cm−1.
Degradation and Aminolysis of Dendritic Comblike Copoly-
mers. Dried DC2 copolymer (30 mg) was dissolved in 3.0 mL of
THF under nitrogen, and about 2.0 μL of Bu3P was added to the
solution at ambient temperature. The mixture was stirred overnight,
diluted with THF and subjected to GPC analysis. Molecular weight
and polydispersity of degraded (PEG)x(PCL)y comblike copolymer
were Mn,LS = 17900, PDI = 1.36.
Aminolysis was used to prepare multithiol-functionalized
(PEG)m(PCL)n(SH)o copolymer. Under nitrogen, 0.50 g of DC2
copolymer was dissolved in 10 mL of THF, and followed by slow
addition of a solution of 10-fold excess of hydrazine in THF. After
stirring for 2 h, the mixture was concentrated and precipitated, and
DC2-SH copolymer was isolated as white powders. Mn,LS = 79600,
PDI = 1.12.
Synthesis of AmBnCo Dendritic Toothbrushlike Copolymers
by Chain Extension Polymerization. In a typical polymerization
(run 1 of Table 2), DC2 copolymer (0.50 g, 34.5 μmol CTA), St (0.72
g, 6.9 mmol), AIBN (1.1 mg, 6.7 μmol) were added to a Schlenk tube,
and toluene was added until the total volume was 4.6 mL. After
degassing with bubbled nitrogen for 20 min, the mixture was
polymerized at 80 °C for 12 h. The polymerization solution was
precipitated into methanol thrice, and 0.643 g (19.9% conversion) of
(PEG)m(PCL)n(PSt)o copolymer was isolated. Mn,LS = 105100, PDI =
1.06. Other dendritic toothbrushlike copolymers were synthesized and
purified according to similar procedures.
(PEG)m(PCL)n(PMMA)o Copolymer. 1H NMR (CDCl3): δ 7.96
(PhH of terminal PhC(S)S), 6.2−7.7 (ArH of St-PEG unit and PhH
of terminal PhC(S)S), 4.50 and 4.30 (CH2O), 4.06 (CH2O of
PCL), 3.88 (CH of MI-PCL unit), 3.65 (CH2CH2O of PEG), 3.60
(CH3O of PMMA and CH2N of MI-PCL unit), 3.38 (CH3O of PEG),
0.8−3.0 (CH, CH2 and CH3 originating from RAFT agent, maleimidic
and styrenic units, and MMA unit). FT-IR (KBr): 3448, 2946, 2870,
1732, 1703, 1638, 1560, 1459, 1398, 1352, 1275, 1245, 1194, 1152,
1099, 1038, 991, 954, 845, 749 cm−1.
(PEG)m(PCL)n(PNIPAM)o Copolymer. 1H NMR (CDCl3): δ 7.90
(PhH of terminal PhC(S)S), 6.2−7.7 (ArH of St-PEG unit and PhH
of terminal PhC(S)S), 4.59 (CHS), 4.50 and 4.30 (CH2O), 4.06
(CH2O of PCL), 4.01 (CH of PNIPAM), 3.88 (CH of MI-PCL unit),
3.65 (CH2CH2O of PEG and CH2N of MI-PCL unit), 3.38 (CH3O of
PEG), 0.8−3.0 (CH, CH2 and CH3 originating from RAFT agent,
maleimidic and styrenic units, and NIPAM unit). FT-IR (KBr): 3439,
3293, 3072, 2932, 2871, 1736, 1704, 1647, 1545, 1459, 1389, 1364,
1352, 1279, 1251, 1171, 1101, 1037, 953, 847 cm−1.
Self-Assembly and In Vitro Drug Release from AmBn
Copolymer Aggregates. The copolymer aggregates were prepared
by dialysis method. AmBn copolymer (5.0 mg) was dissolved in 1.0 mL
of DMSO at room temperature, and then the polymer solution was
added dropwise into 9.0 mL of phosphate buffered saline (PBS, pH
7.4, 50 mM) solution under vigorous stirring. Two hours later, the
solution was transferred into dialysis membrane tubing (MWCO
3500) and dialyzed against PBS solution for 50 h to completely
remove the organic solvent. The solution of copolymer aggregates was
stored at 4 °C before measurement, and the size and morphology of
aggregates were determined by DLS and TEM, respectively.
A similar procedure was used to prepare doxorubicin (DOX)-
loaded aggregates. In a typical run, AmBn copolymer (10.0 mg) and
DOX hydrochloride (2.0 mg) were dissolved in 1.0 mL of DMSO, and
followed by addition of about 0.6 mg of triethylamine. The mixed
solution was added dropwise to 19.0 mL of PBS solution (pH 7.4, 50
mM). After stirring for an additional 4 h, the solution was dialyzed
against PBS solution for 50 h (MWCO 3500). The amount of DOX
was determined using fluorescence (FLS920) measurement (excitation
at 480 nm and emission at 560 nm). The drug loading capacity (DLC)
and drug loading efficiency (DLE) of aggregates were determined by
fluorescence analysis. In a typical run for drug release, two portions of
DOX-loaded copolymer aggregates in PBS solution (4.0 mL, pH 7.4)
were put into a dialysis bag (MWCO 5000), which were then
immersed into 20 mL of (a) PBS solution (50 mM, pH 7.4) with 10
mM DTT or (b) normal PBS solution (50 mM, pH 7.4) at 37 °C. At
predetermined time intervals, the drug-release solution was changed,
and the amount of DOX released from aggregates was measured by
fluorescence measurement (excitation at 480 nm) at room temper-
ature. All release experiments were performed in triplicate.
Characterization. Apparent number-average molecular weight
(Mn,GPC) and polydispersity (PDI) of St-PEG and MI-PCL macro-
monomers were measured on a Waters 150-C GPC using three
Ultrastyragel columns (pore size 50, 100, and 1000 nm) with 10 μm
bead size at 35 °C. THF was used as an eluent at a flow rate of 1.0
mL/min, and samples were calibrated using PMMA standard samples.
Gel permeation chromatography with multiple angle laser scattering
detection (GPC-MALLS) systems was used to determine absolute
(PEG)m(PCL)n(PSt)o Copolymer. 1H NMR (CDCl3): δ 7.86 (PhH of
terminal PhC(S)S), 6.2−7.7 (PhH of PSt, ArH of St-PEG unit and
PhH of terminal PhC(S)S), 4.6−5.1 (CHS), 4.50 and 4.30 (CH2O),
4.06 (CH2O of PCL), 3.88 (CH of MI-PCL unit), 3.65 (CH2CH2O of
PEG and CH2N of MI-PCL unit), 3.38 (CH3O of PEG), 0.8−3.0
(CH, CH2 and CH3 originating from RAFT agent, maleimidic and
styrenic units). FT-IR (KBr): 3447, 3081, 3059, 3025, 2923, 2868,
1774, 1737, 1701, 1602, 1560, 1493, 1454, 1398, 1352, 1299, 1280,
1251, 1146, 1105, 1031, 952, 848, 758, 700 cm−1.
(PEG)m(PCL)n(PtBA)o Copolymer. 1H NMR (CDCl3): δ 7.90 (PhH
of terminal PhC(S)S), 6.2−7.7 (ArH of St-PEG unit and PhH of
terminal PhC(S)S), 4.64 (CHS), 4.50 and 4.30 (CH2O), 4.06
(CH2O of PCL), 3.88 (CH of MI-PCL unit), 3.65 (CH2CH2O of PEG
and CH2N of MI-PCL unit), 3.38 (CH3O of PEG), 0.8−3.0 (CH, CH2
and CH3 originating from RAFT agent, maleimidic and styrenic units,
and tBA unit). FT-IR (KBr): 3435, 2933, 2868, 1731, 1705, 1638,
1561, 1459, 1394, 1368, 1350, 1256, 1154, 1100, 1036, 953, 846, 751
cm−1.
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number-average molecular weight (Mn,LS), polydispersity and solution
viscosity of various copolymers. GPC was conducted in THF at 35 °C
with a flow rate of 1.0 mL/min. Three TSK-GEL H-type columns
(pore size 15, 30, and 200 Å, with molecular weight range of 100−
1000, 300−20000, and 5000−400000 g/mol, respectively) with 5 μm
bead size were used. Detection consisted of a RI detector (Optilab
rEX), a multiangle (14−145°) laser light scattering (MALLS) detector
(DAWN HELEOS) with the He−Ne light wavelength at 658.0 nm,
and online viscosity detector (ViscoStar). The refractive index
increment dn/dc for samples were measured off-line by Optilab rEX
refractive index detector (λ = 658 nm) at 25 °C using a series of
different concentration solutions. Data were collected and processed
by use of ASTRA software from Wyatt Technology, and molecular
weights were determined by the triple detection method. The intrinsic
viscosity of copolymer solutions in THF was measured using a
viscosimetric detector connected to GPC system at 35 °C. 1H and 13
C
NMR spectra were recorded on a Varian spectrometer at 25 °C using
CDCl3 as a solvent. Fourier transform infrared (FT-IR) spectra were
recorded on a Perkin-Elmer 2000 spectrometer using KBr disks. C, H,
N, and S were determined by combustion followed by chromato-
graphic separation and thermal conductivity detection using a Carlo-
Erba EA 1110CHNO-S elemental analyzer. Differential scanning
calorimetry (DSC) analysis was performed under nitrogen atmosphere
using a SDT 2960 Simultaneous DSC−TGA of TA Instruments with
heating rate of 10 °C/min. Dynamic light scattering (DLS)
measurements were carried out at 25 °C using Zetasizer Nano-ZS
from Malvern Instruments equipped with a 633 nm He−Ne laser
using backscattering detection, and the micellar solutions were filtered
through a 450 nm syringe filter before measurements. Fluorescence
spectroscopy was recorded at 25 °C on a FLS920 fluorescence
spectrometer. Transmission electron microscopy (TEM) images were
obtained through a Hitachi H-600 electron microscope.
1
Figure 1. H NMR spectra of MI-PCL macromonomers synthesized
by runs 1−4 of Table S1, Supporting Information.
RESULTS AND DISCUSSION
■
This study aimed at versatile synthesis and properties of
disulfide-functionalized dendritic comblike and toothbrushlike
copolymers. St-PEG and MI-PCL, as monomer pairs with
strong tendency for alternating copolymerization, were
subjected to RAFT copolymerization in the presence of ACP
to form AmBn-type dendritic comblike copolymers, and
AmBnCo-type dendritic toothbrushlike copolymers were gen-
erated by a subsequent chain extension polymerization. The
Figure 2. GPC traces of St-PEG (a) and MI-PCL (b−e) macro-
monomers. Apparent Mn and PDI values: 1570, 1.04 (a); 1990, 1.10
(b); 2920, 1.13 (c); 5260, 1.14 (d); 8090, 1.18 (e).
Synthesis of Dendritic Comblike Copolymers by RAFT
SCVP. RAFT copolymerization in the presence of a polymer-
izable RAFT agent allows for one-pot synthesis of segmented
hyperbranched copolymers with variable degree of branching
and CTA functionality (f CTA).41−43 A disulfide-linked RAFT
agent 2-((2-(acryloyloxy)ethyl)disulfanyl)ethyl 4-cyano-4-
(phenylcarbonothioylthio)pentanoate (ACP) was first synthe-
sized by esterification between 2-((2-hydroxyethyl)disulfanyl)-
ethyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate and
acrylic acid, which acted as a versatile core reagent to generate
multicleavable dendritic comblike copolymers. Unlike normal
segmented hyperbranched copolymers with long linear
branches,38−43 the dendritic comblike copolymers obtained
herein had branches composed of comblike copolymers with
alternating PEG and PCL grafts and multiple disulfide
functionalities in branching points, which enabled versatile
topological transformations via postpolymerization and post-
modification.
1
resultant copolymers were characterized by H NMR, GPC-
MALLS, DSC, and viscosity measurement, and DOX-loading
and release properties of typical AmBn copolymers were
investigated as well.
Synthesis of MI-PCL Macromonomers. MI-PCL was
synthesized by two step reactions involving (a) ROP of CL
initiated with 4-(2-hydroxyethyl)-10-oxa-4-aza-tricyclo-
[5.2.1.02,6]dec-8-ene-3,5-dione (HTD) and (b) a subsequent
deprotection at 120 °C to release the maleimide ring (Table S1,
Supporting Information). In 1H NMR spectra (Figure 1),
characteristic signals of terminal group were noted at δ 6.76
(CHCH), 4.24 (NCH2CH2O), 3.80 (CH2N) and 3.66
(terminal CH2OH), and signals of CH2 in PCL segment
appeared at δ 4.06 (CH2O), 2.31 (CH2CO), 1.65 and 1.40
(CH2CH2CH2CH2O). The polymerization degree (DP) of MI-
PCL could be determined by equation DPNMR = I2.31/I6.76
where I meant the integrated peak area. The molecular weights
determined by 1H NMR (Mn,NMR) were in the range of 1080−
5070, which agreed well with the expected values (Mn,th). The
GPC traces exhibited monomodal distribution with polydisper-
sity in the range of 1.10−1.18 (Figure 2). These results revealed
the two-step strategy could afford the target MI-PCL
macromonomers with well-controlled molecular weight.
ACP and equimolar feed ratio of St-PEG (Mn,NMR = 870, PDI
= 1.04) and MI-PCL (Mn,NMR = 1080, 1580 or 5070)
macromonomers were subjected to RAFT SCVP to generate
disulfide-functionalized AmBn dendritic comblike copolymers
1
(DC1-DC8 of Table 1). In H NMR spectra of typical AmBn
copolymers (Figure 3), the characteristic signals of aromatic
protons originating from ACP were noted at δ 7.98 (2H of
PhH, terminal PhC(S)S) and 6.2−7.8 (ArH and other PhH),
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decrease. The extended reaction time generally increased
molecular weight of the resultant copolymers due to enhanced
monomer conversion and number of branches. By comparing
the results as listed in runs 2 and 3 of Table 1, it can be seen
that the molecular weight increased from 78 300 (DC2) to 136
000 (DC3) as monomer conversion varied from 35.2% (t = 24
h) to 50.8% (t = 48 h), while the GPC trace of DC3 obtained at
a longer time exhibited notably broadened molecular weight
distribution although the polydispersity remained low (PDI =
1.21, Figure 4). The gradually broadened distribution with
Figure 4. GPC traces (normalized weight distribution) of AmBn
dendritic comblike copolymers (DC1−DC3) and normal comblike
copolymer (C1).
extended time was possibly originated from reduced reactivity
of polymer radicals and increased side reactions involving
irreversible termination, and the latter was prone to result in
partial loss of CTA functionality.41
Figure 3. 1H NMR spectra of typical AmBn dendritic comblike
copolymers.
the signals of CH2 protons in PCL segment appeared at δ about
4.06, 2.31, 1.65 and 1.40, and the signals in PEG segment
appeared at δ 3.65 (CH2CH2O) and 3.38 (CH3O). Therefore,
number-average dithiobenzoate functionality could be calcu-
Besides, the effects of monomer concentration and chain
length of MI-PCL on the copolymerization were also
investigated. The results in various runs using same feed ratio
revealed that both total monomer conversion and ACP
conversion were remarkably decreased as MI-PCL concen-
tration was reduced from 0.20 (runs 1−3 of Table 1) to 0.05
mol/L (runs 4−8 of Table 1), which could be attributed to
reduced rate of radical copolymerization. By comparing the
results in runs 4 and 8 of Table 1, it was found that the
copolymerization using high-molecular-weight MI-PCL
(DPNMR = 43.2) was liable to give DC8 copolymer with
lowered CTA functionality and number of grafted chains,
suggesting the decreased reactivity of macromonomer with
enhanced chain length.
lated by equation f CTA = Mn,LS × I7.98/(MWCTA × I7.98
+
M
n,NMR(St-PEG) × I3.65/(2DPPEG) + Mn,NMR(MI-PCL) × I4.06
/
(DPPCL − 1)), and number-average segment numbers of PEG
(m) and PCL (n) grafts per copolymer could be deduced by
equations m = f CTA × I3.65/(2DPPEG × I7.98), and n = fCTA
×
I
4.06/((DPPCL − 1) × I7.98), in which Mn,LS was number-average
molecular weight of AmBn copolymer determined by GPC-
MALLS, DPPEG and DPPCL were polymerization degrees of St-
PEG and MI-PCL determined by 1H NMR, Mn,NMR was
1
molecular weight determined by H NMR, and MWCTA was
molecular weight of ACP or CPDB.
The results as listed in Table 1 indicated that RAFT SCVP
could afford dendritic comblike copolymers with intrinsic
viscosity in the range of 9.2−26.5 mL/g and average CTA
functionality up to 7.5. The similar segment numbers of PEG
and PCL grafts in each copolymer revealed the presence of
alternating pendent chains. Monomodal distribution was
usually noted in GPC traces, and their polydispersity indices
were relatively low (PDI = 1.10−1.32, Figure 4), indicating the
target dendritic comblike copolymers were successfully
obtained by RAFT copolymerization.
Meanwhile, a normal AmBn comblike copolymer lack of
disulfide linkage (C1, Mn,LS = 22 500, PDI = 1.22) was
synthesized by RAFT copolymerization of St-PEG and MI-PCL
(Mn,NMR = 1080) mediated by 2-(2-cyanopropyl) dithioben-
zoate (CPDB). C1 copolymer was used as a “linear” analogue
AmBn dendritic comblike copolymers with relatively low
polydispersity, multiple disulfide moieties and adjustable
segment numbers of A and B grafts are potentially achieved
under optimized conditions, in which many factors involving
molecular weight of MI-PCL, feed ratio, concentration,
temperature and time can play important roles in RAFT
SCVP. A couple of copolymerization experiments were
performed to reveal their effects on molecular parameters of
dendritic copolymers such as molecular weight, CTA
functionality and segment numbers of grafted chains (Table
1). For copolymerization using MI-PCL with fixed polymer-
ization degree, both Mn,LS and segment numbers of m and n
increased with increasing feed ratio of macromonomer to ACP
(x), while the corresponding f CTA values were liable to
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of dendritic comblike copolymer to investigate the influence of
macromolecular architecture on physicochemical properties.
Degradation and Aminolysis of Dendritic Comblike
Copolymers. The presence of multiple disulfide moieties in
AmBn dendritic comblike copolymers enables topological
transformations via degradation and coupling.20 For instance,
the cleavage of disulfide functionalities in AmBn copolymer
upon reductive stimulus such as addition of excess Bu3P and
DTT generates degraded thiol-bearing AxBy-type comblike
copolymer (a); Multithiol-functionalized AmBn(SH)o copoly-
mer is obtained by aminolysis or reduction of terminal
dithiobenzoate moieties, which can be further converted into
AxBy-type comblike copolymer (b) with at least two thiol
functionalities via reduction-triggered degradation (Scheme 2).
Figure 5. GPC traces (normalized weight distribution) of DC2
Scheme 2. Preparation of Thiol-Functionalized AmBn-Type
Dendritic Comblike Copolymer (AmBn(SH)o) and Degraded
AxBy-Type Comblike Copolymers (a and b) via Aminolysis
and Reduction-Triggered Topological Transformation
copolymer before (a, Mn,LS = 78300, PDI = 1.10) and after (b, Mn,LS
=
17900, PDI = 1.36) treatment with Bu3P, in which the dash dot line
was fitted curve.
Synthesis of AmBnCo Dendritic Toothbrushlike Co-
polymers. Postpolymerization involving controlled radical
polymerization and ring-opening polymerization has a great
potential in formation of novel macromolecular architectures.
In this study, AmBn dendritic comblike copolymers were
converted into AmBnCo-type dendritic toothbrushlike copoly-
mers as hydrophobic polystyrene (PSt), poly(tert-butyl
acrylate) (PtBA) and poly(methyl methacrylate) (PMMA),
and hydrophilic poly(N-isopropylacrylamide) (PNIPAM) seg-
ments were grown from the “surface” of AmBn copolymers via
RAFT process.
DC2 (Mn,LS = 78300, f CTA = 5.4) was chosen as a typical
macro CTA to mediate chain extension polymerization of vinyl
1
monomers (Table 2). In H NMR spectra of the isolated
AmBnCo copolymers (Figure 6), the characteristic signals of
These copolymers can oxidize into high-molecular-weight
comblike and dendritic copolymers owing to the high reactivity
of thiol moieties. In this study, (PEG)m(PCL)n copolymer
(DC2, Mn,LS = 78300) was chosen as a typical sample to
perform cleavage and aminolysis. The reduction using excess
Bu3P afforded degraded (PEG)x(PCL)y samples composed of a
series of comblike copolymers with different numbers of
“branches”, evident from significantly decreased molecular
weight (Mn,LS = 17900) and broadened molecular weight
distribution (Figure 5). The gradual degradation upon
reductive stimulus also confirmed the branched architecture
of dendritic comblike copolymers. Meanwhile, aminolysis of
Figure 6. 1H NMR spectra of (PEG)m(PCL)nCo dendritic tooth-
brushlike copolymers.
each segment were noted at δ 6.2−7.2 (PhH of PSt), 3.60
(CH3O of PMMA), 2.23 (CH of PtBA), 1.44 (CH3 of PtBA),
4.01 (CH of PNIPAM), 4.06 (CH2O of PCL), and 3.65
(CH2CH2O of PCL), and signals of terminal groups appeared
at δ 7.8−8.0 (terminal PhH of PhC(S)S moieties), 4.6−5.1
(CHS of terminal St unit), and about 4.6 (CHS of terminal tBA
and NIPAM units). The signals at 4.8−5.1 ppm corresponding
to CHS (terminal comonomer unit) of AmBn copolymer
completely disappeared, and new signals originating from CHS
of terminal tBA and NIPAM units were almost quantitatively
DC2 gave thiol-terminated DC2-SH copolymer (Mn,LS
=
79600), which was used for drug loading and release as
described later.
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noted in 1H NMR spectra, revealing that all the dithiobenzoate
moieties of AmBn copolymer had participated in the RAFT
process. Therefore, the average number of C segment was equal
to the CTA functionality of AmBn copolymer (o ≈ 5.4). By
comparing the integrated peak areas of these protons, Mn,NMR
values of various copolymers were obtained. Their Mn,LS and
Mn,NMR values were comparable, and both agreed well with
theoretical results expected from Mn,LS of macro CTA and
monomer conversion. The resultant AmBnCo copolymers
exhibited roughly symmetric distribution in GPC traces, and
their polydispersity indices were in the range of 1.06−1.13
(Figure 7). These results indicated RAFT chain extension
Figure 7. GPC traces (normalized weight distribution) of DC2 (a)
and (PEG)m(PCL)nCo (C = PSt (b), PtBA (c), PMMA (d), and
PNIPAM (e)) copolymers.
Figure 8. DSC traces of St-PEG (a), MI-PCL (b, DP = 8.2), DC2 (c),
C1 (d), and (PEG)m(PCL)nCo copolymers (C = PSt (e), PtBA (f),
PMMA (g), and PNIPAM (h)).
polymerization was highly efficiently performed to give AmBnCo
dendritic copolymers. The facile synthesis potentially endows
the target AmBnCo terpolymers with a wide range of chain
length and chemical composition of C segment, and the
number of C segment is theoretically equal to f CTA of the
macro CTA if all the dithiobenzoate functionalities are
activated and side reactions resulting in partial loss of chain
radicals are absent.
remarkably reduced melting peaks and broadened melting
range in dendritic comblike copolymer.
AmBnCo dendritic toothbrushlike copolymers have more
complex chain relaxation and melting processes than AmBn
dendritic comblike copolymers due to their different
architectures. In DSC traces, (PEG)m(PCL)n(PtBA)o showed
a notable Tg at 16.9 °C, and other AmBnCo samples only
exhibited a relatively weak Tg at about 62.2 (C = PSt), 85.3 (C
= PMMA) and 116.1 °C (C = PNIPAM). These observations
were in accordance with our previous results,73−75 in which the
Tg values of (PM)m multiarm star and linear PM with same Mn
values decreased in the order linear PM > star-shaped (PM)m >
cleaved PM arm. Interestingly, multiple melting peaks were
normally noted in DSC traces of various AmBnCo samples, and
the melting range could cover a wide range within 17−75 °C.
This phenomenon could be primarily ascribed to the restricted
chain movement during crystallization originating from unique
architecture of AmBnCo terpolymers. With the introduction of C
segments, the resultant dendritic toothbrushlike copolymers
possessed a loose out layer comprising C segments and high-
density inner layer with overpacking PEG and PCL grafts, and a
wide range of microdomains with different types of crystalline
and noncrystalline regions and variable degree of crystallinity
could be formed, resulting in significantly broadened melting
range in DSC traces. The detailed restricted crystallization
behaviors of AmBn and AmBnCo dendritic copolymers are in
progress in our laboratory.
DSC Analysis of Dendritic Copolymers. DSC measure-
ment was performed to investigate the effects of macro-
molecular architecture on chain relaxation and melting
behaviors (Table S2, Supporting Information). Macromono-
mers were of melting peaks (Tm) at 29.5 °C (St-PEG), 32.7 and
42.6 °C (MI-PCL), respectively. AmBn dendritic comblike
copolymer (DC2) had weak glass transitions (Tg) at 47.2 and
54.5 °C and melting range within 15.8−44.9 °C with two
melting peaks at 19.0 and 26.8 °C (Figure 8c). Normal AmBn
comblike copolymer (C1) exhibited obvious glass transitions at
53.6 and 67.3 °C and melting range within 17.8−43.8 °C with
melting peak at 33.0 °C (Figure 8d). Besides different chain
length of poly(styrene-alt-maleimide) backbones, the branching
effect could account for their different thermal properties.
Although both DC2 and C1 possessed same PEG and PCL side
chains, comblike copolymer C1 had longer linear backbone,
while dendritic comblike copolymer DC2 was of branched
backbones with a couple of comonomer units in each branch,
and thus dendritic comblike copolymer could perform the
chain relaxation at reduced temperature. Meanwhile, the
presence of more compact branched structures in DC2 was
liable to form multiple restricted crystalline and noncrystalline
regions in which the folding and rearrangement of PEG and
PCL chains were more or less disturbed, resulting in
Solution Behavior of Dendritic Copolymers. Parame-
ters involving Mark−Houwink-Sakurada (MHS) exponent (α)
and contracting factor (g′) can be used to describe different
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solution properties between branched polymers and their linear
analogues. The MHS exponent of branched polymer is smaller
than that of its linear counterpart, and the branching effect can
be expressed by the contracting factor defined as the intrinsic
viscosity ratio between branched and linear samples with same
Mw values.39−41 Herein we used [η]dc/[η]c to illustrate the
branching effect, in which [η]dc and [η]c were weight-average
intrinsic viscosities of dendritic comblike copolymers and
normal comblike copolymers with same molecular weights.
Three typical AmBn (A = PEG, B = PCL) copolymers
originating from same macromonomers (DPPEG = 16, DPPCL
= 8.2) were subjected to viscosity measurements, and their
Mark−Houwink−Sakurada plots over a selected common
molecular weight range are listed in Figure 9. The MHS
Figure 10. Mark−Houwink-Sakurada plots of DC2 (a) and
(PEG)m(PCL)nCo copolymers (C = PSt (b), PtBA (c), PMMA (d),
and PNIPAM (e)).
copolymer aggregates. DLS results revealed that various
copolymers were liable to self-assemble into aggregates with
bimodal distribution. DC2 formed blank aggregates with peak
diameters (Dpeak) of 22.8 and 178.1 nm, and its DOX-loaded
copolymer aggregates had Dpeak values of 18.7 and 232.3 nm
(Figure 11a). Although DC2-SH formed bimodal-distributed
Figure 9. Mark−Houwink−Sakurada plots of normal comblike
copolymer (C1) and typical dendritic comblike copolymers (DC2
and DC3).
equations of various copolymers obtained in Table 1 were
0.338
determined to be [η]dc = 0.221Mw0.368 (DC2) and 0.296Mw
(DC 3), and [η]c = 0.195Mw
0.395
(C1), and the [η]dc/[η]c
values were calculated to be 0.858 (a) and 0.772 (b). Dendritic
comblike copolymer was of reduced α values and smaller
intrinsic viscosities than normal comblike copolymer with same
molecular weight, further confirming the presence of more
compact “hyperbranched” structure in dendritic copolymers.
Dendritic toothbrushlike copolymers may exhibit complex
solution properties due to their unique architectures comprising
compact “hyperbranched” inner layer and loose “linear” out
layer. Mark−Houwink−Sakurada plots of AmBnCo copolymers
(Figure 10b−e) gave α values in the range of 0.484−0.552, and
the MHS equations of various copolymers were determined to
Figure 11. DLS plots of blank (square) and DOX-loaded (triangle,
Wpolymer:WDOX = 5:1) copolymer aggregates (c = 0.5 mg/mL) in PBS
solution (pH 7.4, 50 mM) at 37 °C.
aggregates (Dpeak = 19.5 and 153.4 nm), its DOX-incorporated
aggregates exhibited a predominant distribution (Dpeak = 263.3
nm) besides a very small peak centering at 45.5 nm (Figure
11b), which revealed the addition of hydrophobic drug could
optimize the self-assembly process. Similarly, bimodal distribu-
tion was noted in DLS plots of C1 aggregates. These results
indicated self-assembly behaviors were affected by a combina-
tion of macromolecular architecture, molecular weight, end
group and even added drugs.
0.552
0.498
be [η] = 0.0327Mw
(b, C = PSt), 0.0697Mw
(c, C =
PtBA), 0.0692Mw0.484 (d, C = PMMA) and 0.0598Mw0.506 (e, C
= PNIPAM). As compared with the MHS equations of linear
polymers [η]l = 0.0129Mw0.740 (PSt), 0.0225Mw0.659 (PtBA) and
0.686
0.0135Mw
(PMMA),41 the MHS exponent and intrinsic
viscosity values of different samples with same Mw values were
found to decrease in the order linear C segment > AmBnCo
dendritic toothbrush copolymer > AmBn dendritic comblike
copolymer. These results revealed that the introduction of C
segments into the surface of dendritic comblike copolymer
could partly decrease the compact degree of dendritic
macromolecules.
DOX-Loading and Release from AmBn Copolymer
Aggregates. Aqueous self-assembly of C1, DC2, and DC2-
SH was performed to prepare blank and DOX-loaded
Owing to the change in macromolecular architecture and
intermolecular interactions, the presence of cleavable disulfide
moieties in dendritic comblike copolymers was liable to damage
the self-assembled structures upon reductive stimulus. In TEM
images of blank DC2 aggregates (Figure S5a, Supporting
Information), both unimolecular micelles (D ≈ 5.0 nm) and
conventional micelles (D ≈ 60 nm) were observed, and the
particle sizes were reasonably smaller than those estimated by
DLS. With addition of 10 mM DTT, nanoparticles
corresponding to unimolecular micelles and aggregates formed
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Table 3. Influence of Architecture and Compositions on Properties of Copolymer Aggregates
a
a
a
b
b
b
c
c
copolymer
D (nm)
PD
Dpeak (nm)
D (nm)
PD
Dpeak (nm)
DLC (%)
DLE (%)
DC2
87.4
90.8
0.585
0.570
0.387
22.8, 178.1
19.5, 153.4
21.3, 154.5
93.3
190.8
36.1
0.650
0.210
0.527
18.7, 232.3
45.5, 263.3
19.0, 227.8
3.32
2.97
3.46
16.6
14.9
17.3
DC2-SH
C1
100.3
a
Cumulant diameter (D), particle size distribution (PD), and peak diameter (Dpeak) of blank copolymer aggregates obtained by DLS analyses.
Various parameters of DOX-loaded copolymer aggregates (5:1). Determined by fluorescence analysis.
b
c
by a couple of copolymers were formed (Figure S5b,
Supporting Information). The remarkably decreased particle
size could be attributed to reduction-triggered destabilization
and rearrangement of copolymer aggregates originating from
the end group effect, decreased molecular weight, and
weakened intermolecular interactions.76−83
DOX loading and release properties of AmBn and AmBn(SH)o
copolymers were investigated to better understand the potential
of copolymer aggregates. The drug loading capacity (DLC) and
drug loading efficiency (DLE) of copolymer aggregates were
determined by fluorescence analysis (Table 3). The DLE value
of DC2 aggregates (DLE = 16.6%) was comparable to that of
C1 aggregates (DLE = 17.3%), and both were slightly higher
than that of DC2-SH aggregates (DLE = 14.9%). The in vitro
drug release of DOX from the copolymer aggregates was
performed in PBS solution (pH 7.4, 50 mM) at 37 °C. The
release rate was liable to decrease in the order DC2-SH > C1 >
DC2 (Figure 12), revealing both macromolecular architecture
thiol-terminated degraded comblike copolymers, and the
change in microenvironment could induce rearrangement and
reaggregation of copolymers due to variable inter and
intramolecular interactions, and thus the encapsulated DOX
could be faster released. With variation of end group and
addition of reductive stimulus, the drug release kinetics could
be tuned in a wide range, revealing the great potential of
dendritic comblike copolymer aggregates for biomedical
application.
CONCLUSION
■
We have demonstrated that the combination of RAFT SCVP
and sequence-controlled copolymerization could be efficiently
used to construct novel multicleavable amphiphilic dendritic
copolymers via one or two step reactions. RAFT copolymeriza-
tion of St-PEG and MI-PCL in the presence of ACP afforded
(PEG)m(PCL)n dendritic comblike copolymers with alternating
PEG and PCL pendent chains and multiple disulfide
functionalities in branching points, and AmBnCo dendritic
toothbrushlike copolymers were generated by a subsequent
chain extension polymerization of vinyl monomers. The
resultant (PEG)m(PCL)n dendritic comblike copolymers had
adjustable molecular weight, relatively low polydispersity,
similar numbers of PEG and PCL grafts, and variable CTA
functionality, evident from 1H NMR and GPC-MALLS
analyses. Reduction-triggered degradation, viscosity measure-
ment and DSC results fully confirmed the branched
architectures of various dendritic copolymers. In vitro drug
release revealed the disulfide-linked copolymer aggregates could
rapidly release the encapsulated doxorubicin when triggered by
10 mM DTT, and the drug release kinetics was remarkably
affected by macromolecular architecture, end group and
reductive stimulus. These reduction-sensitive and biodegrad-
able dendritic copolymer aggregates had a great potential for
biomedical applications. Moreover, the general methodology
can be extended to facile synthesis of numerous dendritic
copolymers with rich chemical composition, tunable segment
length and versatile stimuli-responsive functionality.
Figure 12. In vitro drug release profiles of DOX-loaded copolymer
aggregates in PBS solution (pH 7.4, 50 mM) at 37 °C with or without
addition of 10 mM DTT.
and end group could play important roles in drug release
kinetics. As expected, the cumulative release from DOX-loaded
C1 aggregates in the presence of 10 mM DTT was only slightly
higher than that without DTT, while a faster release from the
dendritic copolymer aggregates upon reductive stimulus was
noted. After 120 h, about 28.4% (DC2), 43.1% (DC2 + DTT),
42.0% (DC2-SH), 53.1% (DC2-SH + DTT), 32.1% (C1), and
33.0% (C1 + DTT) of encapsulated DOX could be efficiently
released from the DOX-loaded copolymer aggregates, and no
burst release behaviors were noted in all cases. These results
indicated the DOX release kinetics was accelerated by the
introduction of reductive stimulus and terminal thiol
functionality, which could be ascribed to dynamic destabiliza-
tion of copolymer aggregates resulting from the change in
molecular weight and end group.76−83 The addition of excess
DTT led to the gradual cleavage of disulfide moiety to form
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic routes to St-PEG and MI-PCL macromonomers,
1
synthesis and DSC data, H and 13C NMR spectra of ACP,
HTD, and macromonomers, IR spectra of ACP and various
polymers, and TEM images of DC2 copolymer aggregates. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ylzhao@suda.edu.cn.
Author Contributions
†These authors contributed equally to this work.
1334
dx.doi.org/10.1021/ma3025283 | Macromolecules 2013, 46, 1325−1336

Macromolecules
Article
(30) Chen, Y. M. Macromolecules 2012, 45, 2619−2631.
(31) Frechet, J. M. J.; Henmi, M.; Gitsov, I.; Aoshima, S.; Leduc, M.
Notes
́
The authors declare no competing financial interest.
R.; Grubbs, R. B. Science 1995, 269, 1080−1083.
(32) Matyjaszewski, K. Macromolecules 2012, 45, 4015−4039.
(33) Rikkou-Kalourkoti, M.; Matyjaszewski, K.; Patrickios, C. S.
Macromolecules 2012, 45, 1313−1320.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (Grants 20874067, 21074081 and
21274096), the Key Project of Chinese Ministry of Education
(No. 209049), and the Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education
Institutions. The authors are grateful for helpful discussions
from Jianzhong Du at Tongji University.
(34) Pugh, C.; Singh, A.; Samuel, R.; Ramos, K. M. B. Macromolecules
2010, 43, 5222−5232.
(35) Muthukrishnan, S.; Jutz, G.; Andre,
E. Macromolecules 2005, 38, 9−18.
́
X.; Mori, H.; Muller, A. H.
̈
(36) Powell, K. T.; Cheng, C.; Wooley, K. L. Macromolecules 2007,
40, 4509−4515.
(37) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T.
P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.;
Rizzardo, E.; Thang, S. H. Macromolecules 1998, 31, 5559−5562.
(38) Wan, W. M.; Pan, C. Y. Macromolecules 2008, 41, 5085−5088.
(39) Wang, Z. M.; He, J. P.; Tao, Y. F.; Yang, L.; Jiang, H. J.; Yang, Y.
L. Macromolecules 2003, 36, 7446−7452.
REFERENCES
(1) Harth, E. M.; Hecht, S.; Helms, B.; Malmstrom, E. E.; Frec
M. J.; Hawker, C. J. J. Am. Chem. Soc. 2002, 124, 3926−3938.
(2) Gillies, E. R.; Jonsson, T. B.; Frec
■
́
het, J.
́
het, J. M. J. J. Am. Chem. Soc.
2004, 126, 11936−11943.
(40) Vogt, A. P.; Sumerlin, B. S. Macromolecules 2008, 41, 7368−
7373.
(3) Gao, C.; Yan, D. Y. Prog. Polym. Sci. 2004, 29, 183−275.
(4) Jin, H. B.; Huang, W.; Zhu, X. Y.; Zhou, Y. F.; Yan, D. Y. Chem.
Soc. Rev. 2012, 41, 5986−5997.
(41) Zhang, C. B.; Zhou, Y.; Liu, Q.; Li, S. X.; Perrier, S.; Zhao, Y. L.
Macromolecules 2011, 44, 2034−2049.
(5) Voit, B. I.; Lederer, A. Chem. Rev. 2009, 109, 5924−5973.
(6) Wurm, F.; Frey, H. Prog. Polym. Sci. 2011, 36, 1−52.
(7) Carlmark, A.; Hawker, C. J.; Hult, A.; Malkoch, M. Chem. Soc.
Rev. 2009, 38, 352−362.
(42) Zhang, M. J.; Liu, H. H.; Shao, W.; Ye, C. N.; Zhao, Y. L.
Macromolecules 2012, 45, 9312−9325.
(43) Han, J.; Li, S. P.; Tang, A. J.; Gao, C. Macromolecules 2012, 45,
4966−4977.
(8) Zagar, E.; Zigon, M. Prog. Polym. Sci. 2011, 36, 53−88.
(9) Konkolewicz, D.; Monteiro, M. J.; Perrier, S. Macromolecules
2011, 44, 7067−7087.
(44) Ida, S.; Terashima, T.; Ouchi, M.; Sawamoto, M. J. Am. Chem.
Soc. 2009, 131, 10808−10809.
(10) Khandare, J.; Calderon, M.; Dagia, N. M.; Haag, R. Chem. Soc.
Rev. 2012, 41, 2824−2848.
(45) Ida, S.; Ouchi, M.; Sawamoto, M. J. Am. Chem. Soc. 2010, 132,
14748−14750.
(11) Li, Z. B.; Kesselman, E.; Talmon, Y.; Hillmyer, M. A.; Lodge, T.
P. Science 2004, 306, 98−101.
(46) Hibi, Y.; Ouchi, M.; Sawamoto, M. Angew. Chem., Int. Ed. 2011,
50, 7434−7437.
(12) Lodge, T. P.; Rasdal, A.; Li, Z. B.; Hillmyer, M. A. J. Am. Chem.
Soc. 2005, 127, 17608−17609.
(47) Nakatani, K.; Ogura, Y.; Koda, Y.; Terashima, T.; Sawamoto, M.
J. Am. Chem. Soc. 2012, 134, 4373−4383.
(13) Moughton, A. O.; Hillmyer, M. A.; Lodge, T. P. Macromolecules
2012, 45, 2−19.
(48) Satoh, K.; Saitoh, S.; Kamigaito, M. J. Am. Chem. Soc. 2007, 129,
9586−9587.
(14) Fukae, K.; Terashima, T.; Sawamoto, M. Macromolecules 2012,
45, 3377−3386.
(49) Mizutani, M.; Satoh, K.; Kamigaito, M. J. Am. Chem. Soc. 2010,
132, 7498−7507.
(15) Gao, H. F.; Matyjaszewski, K. Prog. Polym. Sci. 2009, 34, 317−
(50) Satoh, K.; Matsuda, M.; Nagai, K.; Kamigaito, M. J. Am. Chem.
Soc. 2010, 132, 10003−10005.
350.
(16) Altintas, O.; Vogt, A. P.; Barner-Kowollik, C.; Tunca, U. Polym.
Chem. 2012, 3, 34−45.
(17) Bapat, A. P.; Roy, D.; Ray, J. G.; Savin, D. A.; Sumerlin, B. S. J.
Am. Chem. Soc. 2011, 133, 19832−19838.
(51) Li, Z. L.; Li, L.; Deng, X. X.; Zhang, J. J.; Dong, B. T.; Du, F, S.;
Li, Z. C. Macromolecules 2012, 45, 4590−4598.
(52) Chen, G. Q.; Wu, Z. Q.; Wu, J. R.; Li, Z. C.; Li, F. M.
Macromolecules 2000, 33, 232−234.
(18) Syrett, J. A.; Haddleton, D. M.; Whittaker, M. R.; Davis, T. P.;
Boyer, C. Chem. Commun. 2011, 47, 1449−1451.
(19) Zhang, Q.; Li, G. Z.; Becer, C. R.; Haddleton, D. M. Chem.
Commun. 2012, 48, 8063−8065.
(53) Pfeifer, S.; Lutz, J.-F. J. Am. Chem. Soc. 2007, 129, 9542−9543.
(54) Badi, N.; Lutz, J.-F. Chem. Soc. Rev. 2009, 38, 3383−3390.
(55) Berthet, M.-A.; Zarafshani, Z.; Pfeifer, S.; Lutz, J.-F. Macro-
molecules 2010, 43, 44−50.
(56) Lutz, J.-F.; Schmidt, B. V. K. J.; Pfeifer, S. Macromol. Rapid
Commun. 2011, 32, 127−135.
(57) Chan-Seng, D.; Zamfir, M.; Lutz, J.-F. Angew. Chem., Int. Ed.
2012, 51, 12254−12257.
(58) Moughton, A. O.; Sagawa, T.; Gramlich, W. M.; Seo, M.; Lodge,
T. P.; Hillmyer, M. A. Polym. Chem. 2013, 4, 166−173.
(59) Zhu, H.; Deng, G. H.; Chen, Y. M. Polymer 2008, 49, 405−411.
(60) Deng, G. H.; Chen, Y. M. J. Polym. Sci., Part A: Polym. Chem.
2009, 47, 5527−5533.
(61) Xia, N.; Zhang, G. L.; Li, T.; Wang, W.; Zhu, H.; Chen, Y. M.;
Deng, G. H. Polymer 2011, 52, 4581−4589.
(62) Li, S. X.; Ye, C. N.; Zhao, G. D.; Zhang, M. J.; Zhao, Y. L. J.
Polym. Sci., Part A: Polym. Chem. 2012, 50, 3135−3148.
(63) Zhao, Y. L.; Jiang, J.; Liu, H. W.; Chen, C. F.; Xi, F. J. Polym. Sci.,
Part A: Polym. Chem. 2001, 39, 3960−3966.
(20) Jiang, X.; Zhang, M. J.; Li, S. X.; Shao, W.; Zhao, Y. L. Chem.
Commun. 2012, 48, 9906−9908.
(21) Ye, C. N.; Zhao, G. D.; Zhang, M. J.; Du, J. Z.; Zhao, Y. L.
Macromolecules 2012, 45, 7429−7439.
(22) Sheiko, S. S.; Sumerlin, B. S.; Matyjaszewski, K. Prog. Polym. Sci.
2008, 33, 759−785.
(23) Lee, H. I.; Pietrasik, J.; Sheiko, S. S.; Matyjaszewski, K. Prog.
Polym. Sci. 2010, 35, 24−44.
(24) Iha, R. K.; Wooley, K. L.; Nystrom, A. M.; Burke, D. J.; Kade, M.
̈
J.; Hawker, C. J. Chem. Rev. 2009, 109, 5620−5686.
(25) Xia, Y.; Boydston, A. J.; Grubbs, R. H. Angew. Chem., Int. Ed.
2011, 50, 5882−5885.
(26) Xia, Y.; Li, Y. J.; Burts, A. O.; Ottaviani, M. F.; Tirrell, D. A.;
Johnson, J. A.; Turro, N. J.; Grubbs, R. H. J. Am. Chem. Soc. 2011, 133,
19953−19959.
(27) Feng, C.; Li, Y. J.; Yang, D.; Hu, J. H.; Zhang, X. H.; Huang, X.
Y. Chem. Soc. Rev. 2011, 40, 1282−1295.
(64) Zhao, Y. L.; Zhang, J. M.; Jiang, J.; Chen, C. F.; Xi, F. J. Polym.
Sci., Part A: Polym. Chem. 2002, 40, 3360−3366.
(65) Zhao, Y. L.; Chen, C. F.; Xi, F. J. Polym. Sci., Part A: Polym.
Chem. 2003, 41, 2156−2165.
(28) Li, A.; Li, Z.; Zhang, S. Y.; Sun, G. R.; Policarpio, D. M.;
Wooley, K. L. ACS Macro Lett. 2012, 1, 241−245.
(29) Xu, Y. Y.; Bolisetty, S.; Ballauff, M.; Muller, A. H. E. J. Am.
̈
Chem. Soc. 2009, 131, 1640−1641.
(66) Deng, G. H.; Chen, Y. M. Macromolecules 2004, 37, 18−26.
1335
dx.doi.org/10.1021/ma3025283 | Macromolecules 2013, 46, 1325−1336

Macromolecules
Article
(67) Liu, Q. C.; Chen, Y. M. Macromol. Chem. Phys. 2007, 208,
2455−2462.
(68) Konkolewicz, D.; Gray-Weale, A.; Perrier, S. J. Am. Chem. Soc.
2009, 131, 18075−18077.
(69) Konkolewicz, D.; Poon, C. K.; Gray-Weale, A.; Perrier, S. Chem.
Commun. 2011, 47, 239−241.
(70) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.;
Cornelissen, J. J. L. M.; Velonia, K. J. Am. Chem. Soc. 2005, 127, 2966−
2973.
(71) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L.
Macromolecules 2001, 34, 2248−2256.
(72) Tao, L.; Liu, J. Q.; Tan, B. H.; Davis, T. P. Macromolecules 2009,
42, 4960−4962.
(73) Zhao, Y. L.; Cai, Q.; Jiang, J.; Shuai, X. T.; Bei, J. Z.; Chen, C. F.;
Xi, F. Polymer 2002, 43, 5819−5825.
(74) Zhao, Y. L.; Shuai, X. T.; Chen, C. F.; Xi, F. Chem. Mater. 2003,
15, 2836−2843.
(75) Zhao, Y. L.; Chen, Y. M.; Chen, C. F.; Xi, F. Polymer 2005, 46,
5808−5819.
(76) Kujawa, P.; Watanabe, H.; Tanaka, F.; Winnik, F. M. Eur. Phys. J.
E. 2005, 17, 129−137.
(77) Koga, T.; Tanaka, F.; Motokawa, R.; Koizumi, S.; Winnik, F. M.
Macromolecules 2008, 41, 9413−9422.
(78) Xu, J.; Tao, L.; Boyer, C.; Lowe, A. B.; Davies, T. P.
Macromolecules 2011, 44, 299−312.
(79) Du, J. Z.; Willcock, H.; Patterson, J. P.; Portman, I.; O’Reilly, R.
K. Small 2011, 7, 2070−2080.
(80) Zhu, Y. Q.; Liu, L.; Du, J. Z. Macromolecules 2013, 46, 194−203.
(81) Klaikherd, A.; Nagamani, C.; Thayumanavan, S. J. Am. Chem.
Soc. 2009, 131, 4830−4838.
(82) Meng, F. H.; Hennink, W. E.; Zhong, Z. Y. Biomaterials 2009,
30, 2180−2198.
(83) Khorsand Sourkohi, B.; Schmidt, R.; Oh, J. K. Macromol. Rapid
Commun. 2011, 32, 1652−1657.
1336
dx.doi.org/10.1021/ma3025283 | Macromolecules 2013, 46, 1325−1336