Dendritic poly(benzyl ether)-b-poly(N-vinylcaprolactam) block copolymers: Self-organization in aqueous media, thermoresponsiveness and biocompatibility

Article abstract of DOI:10.1002/pola.28897

Four generations of new amphiphilic thermoresponsive linear-dendritic block copolymers (LDBCs) with a linear poly(N-vinylcaprolactam) (PNVCL) block and a dendritic poly(benzyl ether) block are synthesized by atom transfer radical polymerization (ATRP) of N-vinylcaprolactam (NVCL) using dendritic poly(benzyl ether) chlorides as initiators. The copolymers have been characterized by ¹H NMR, FTIR, and GPC showing controlled molecular weight and narrow molecular weight distribution (PDI ≤ 1.25). Their self-organization in aqueous media and thermoresponsive property are highly dependent on the generation of dendritic poly(benzyl ether) block. It is observed for the LDBCs that the self-assembled morphology changes from irregularly spherical micelles, vesicles, rod-like large compound vesicles (LCVs), to the coexistence of spherical micelles and rod-like LCVs, as the generation of the dendritic poly(benzyl ether) increases. The results of a cytotoxicity study using an MTT assay method with L929 cells show that the LDBCs are biocompatible.

Full text of DOI:10.1002/pola.28897

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Dendritic Poly(benzyl ether)-b-poly(N-vinylcaprolactam) Block
Copolymers: Self-Organization in Aqueous Media, Thermoresponsiveness
and Biocompatibility
Gang Tang, Minqi Hu, Fuxi He, Dan You, Yangyang Qian, Yunmei Bi
College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, People’s Republic of China
Correspondence to: Y. Bi (E-mail: yunmeibi@hotmail.com)
Received 1 September 2017; accepted 18 October 2017; published online 00 Month 2017
DOI: 10.1002/pola.28897
ABSTRACT: Four generations of new amphiphilic thermorespon-
sive linear-dendritic block copolymers (LDBCs) with a linear
poly(N-vinylcaprolactam) (PNVCL) block and a dendritic poly(benzyl
ether) block are synthesized by atom transfer radical polymeri-
zation (ATRP) of N-vinylcaprolactam (NVCL) using dendritic pol-
y(benzyl ether) chlorides as initiators. The copolymers have
been characterized by 1H NMR, FTIR, and GPC showing con-
trolled molecular weight and narrow molecular weight distribu-
tion (PDI ꢀ 1.25). Their self-organization in aqueous media and
thermoresponsive property are highly dependent on the gener-
ation of dendritic poly(benzyl ether) block. It is observed for the
LDBCs that the self-assembled morphology changes from
irregularly spherical micelles, vesicles, rod-like large compound
vesicles (LCVs), to the coexistence of spherical micelles and
rod-like LCVs, as the generation of the dendritic poly(benzyl
ether) increases. The results of a cytotoxicity study using an
MTT assay method with L929 cells show that the LDBCs are bio-
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KEYWORDS: biocompatibility; linear-dendritic block copolymers;
poly(N-vinylcaprolactam);
self-assembly;
synthesis;
thermoresponsiveness
applications in drug delivery, nanotechnology, and tissue
engineering.14–18 When an aqueous solution of a thermores-
ponsive polymer is heated above its lower critical solution
temperature (LCST), a macroscopic phase separation occurs.
Poly(N-vinylcaprolactam) (PNVCL) is regarded as second
only to poly(N-isopropylacrylamide) (PNIPAM) the most pop-
ular thermoresponsive polymer.19–21 PNVCL has a LCST in
aqueous solution at temperatures close to physiological tem-
peratures. Apart from that, PNVCL is nonionic, nontoxic, and
biocompatible. In comparison with other studied thermores-
ponsive polymers, PNVCL is not able to form hydrogen
bonds due to the lack of a H-donor in the molecular struc-
ture (unlike, e.g., PNIMAM), which results in lower interac-
tion with biomolecules, higher expected bioacceptability, and
no tendency to crystallization, which may cause poly(2-iso-
propyl-2-oxazoline) to phase separate partly irreversibly. It is
also not poly(ethyleneoxide)-based, which would result in
formation anti-PEO antibodies.22–25 These make PNVCL more
suitable for biomedical applications.26
INTRODUCTION Linear-dendritic block copolymers (LDBCs),
which combine the easy synthesis, availability, and process-
ability of linear polymeric chains with the regular and well-
defined dendritic segments, are particularly attractive due to
their multicomponent constitutions, unique self-assembly
properties, and multivalent characteristics.1–3 Importantly, the
solution and solid state properties of the LDBCs can be tuned
by adjusting the length of the linear chains, generation number
of the dendron, and the chemical structure/polarity of both
blocks. Amphiphilic LDBCs, as a special type of LDBCs, can
self-assemble into various supramolecular structures in aque-
ous solution, which exhibit excellent loading capacity for
hydrophobic small molecules medicine, since most drug mole-
cules are hydrophobic and therefore can be useful in drug
delivery.4–12 In particular, if stimulus-sensitive moieties are
incorporated into the chemical structure of the amphiphilic
LDBCs, the resulting polymeric aggregates may have a con-
trolled and programmed response to an external stimulus.13
Along with all possible stimuli, temperature is an especially
attractive one because the corresponding functional moieties
are comparatively easy to be introduced and fabricated.
The linearly PNVCL-based block copolymers have gained
much impetus in recent years due to the enhanced boost of
the so-called controlled free radical polymerization methods,
such as atom transfer free radical polymerization
Over the past decades, thermoresponsive polymers have
been extensively investigated due to their promising
Additional Supporting Information may be found in the online version of this article.
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(ATRP),27–29 reversible addition fragmentation chain transfer
(RAFT) polymerization,30–34 or organometallic-mediated rad-
ical polymerization.35–37 However, only six recent articles
were concerned on nonlinear block copolymers containing the
PNVCL moiety.38–43 Recently, star-shaped copolymers with
four and six poly(e-caprolactone)-block-poly(N-vinylcaprolac-
tam) S(PCL-b-PNVCL) arms were successfully synthesized by
combining ring opening polymerization of e-caprolactone (CL)
and RAFT polymerization of N-vinylcaprolactam (NVCL).39
Hyperbranched polycaprolactone-click-poly(N-vinylcaprolac-
tam) amphiphilic copolymers was synthesized.40 We have
reported the first preparation of thermoresponsive LDBCs
composed PNVCL and dendritic aromatic polyamides of two
different generations via the RAFT technique, employing den-
dritic chain-transfer agents possessing a single dithiocarba-
mate moiety at the focal point.41 However, the studies on the
self-assembly behavior of higher generations than [G-2] of
PNVCL-based LDBCs still remain largely unexplored.
hexamethyl-1, 4, 8, 11-tetra-azacyclotetradecane (Me6Cy-
clam) was prepared according to literature method.53 1,4-
Dioxane was refluxed on sodium and distilled from sodium
benzophenone. 3,5-Dihydroxybenzyl alcohol was synthesized
according to the method previously reported.54 Isopropanol
was used as received.
Characterizations
1H NMR spectra were recorded on a Bruker DRX-500 spec-
trometer with CDCl3 or D2O as the solvent. The chemical
shifts were calculated relative to TMS. FT-IR spectra were
obtained on a Nicolet AVATAR 360 FT-IR spectrometer.
Molecular weights and molecular weight distributions were
determined using a Waters 2690D gel permeation chromato-
graph equipped a 2410 refractive index detector, Waters
Styragel HR1 THF and HR2 THF columns with THF as eluent
at 40 8C and a flow rate of 0.3 Ml min21. The molecular
weights were calibrated against polystyrene (PS) standards.
DLS measurements were performed with a Zetasizer ZEN
3600 instrument (Malvern, UK) operating at 25–45 8C using
a light scattering apparatus equipped with a He-Ne laser.
The scattering angle was kept at 1738 (backscattering) and
the wave length in the vacuum was set as 633 nm during
the whole experiment. Malvern DTS 6.20 software was used
to analyze the data. Each reported measurement was the
average of three runs. Transmission electron microscopy
(TEM) experiments were carried out on a Hitachi H-600
instrument operating at an acceleration voltage of 80 kV.
In general, the synthesis methods for LDBCs include (i) the
coupling method: coupling of a previously synthesized den-
dron and a monofunctional or oligofunctional linear polymer
chain; (ii) the “chain-first” method: one or two dendrons are
radially grown from a linear polymer; (iii) the “dendron-
first” method: a linear polymer is polymerized from the focal
points of a dendron.1,10 A facile strategy for preparing well-
defined LDBCs is the use of dendrons as macromolecular ini-
tiators for the controlled free radical polymerization of vinyl
ꢀ
monomers. This strategy was pioneered by Frechet and Git-
sov.44–46 The dendron-first approach has proven to be very
useful for the preparation of poly(benzyl ether) based
LDBCs.1
Synthesis of Dendritic Benzyl Alcohols (Gn-OH)
Dendritic benzyl alcohols (Gn-OH) was synthesized according
to the previously published literature54,55 with modification.
Generally, controlling the morphology of self-assembled
aggregates of LDBCs, through the tuning of generation num-
ber of dendritic segment, is of great importance to their
related properties and practical applications.47–50 It has been
reported that some dendrimers display generation-
dependent cytotoxicity.51,52 In this study, a new series of
thermoresponsive amphiphilic linear-dendritic block copoly-
mers (LDBCs) based on PNVCL as a hydrophilic linear block
and four generations of dendritic poly(benzyl ether) as a
hydrophobic block were synthesized by growing a PNVCL
linear arm from the focal point of poly(benzyl ether) den-
drons via ATRP. The effect of the generation number of the
dendritic block and the concentrations of the LDBCs solu-
tions on the thermoresponsive properties and micellar char-
acteristics of these LDBCs in an aqueous phase were
investigated. The cytotoxicity of the LDBCs in vitro was also
evaluated for potential biomedical application.
Synthesis of G1-OH
K2CO3 (13.8 g) was dissolved in 100 mL of DMF and stirred
under nitrogen for 1 h. 3,5-Dihydroxy benzyl alcohol (5.6 g,
40 mmol) was added. Then benzoyl chloride (6.8 mL, 58.6
mmol) was added dropwise to the solution at 65 8C and
stirred for 4 h. The reaction mixture was then poured into
ice-cooled water and the precipitate was collected by filtra-
tion. Crude mixture was purified by a silica column (petro-
leum ether/EtOAc, v/v 5 1.5:1) and the product was
obtained as white solid in 90% yield.
1H NMR (500 MHz, CDCl3, d): 4.62 (d, J 5 6.0 Hz, 2H; CH2O),
5.04 (s, 4H, PhCH2O), 6.55 (t, J 5 2.1 Hz, 1H; CH), 6.63 (d,
J 5 2.1 Hz, 2H; CH), 7.25–7.43 (m, 10H, Ar H).
Synthesis of G2-OH
G2-OH was synthesized by the same general procedure
described for the synthesis of G1-OH. From G1-Cl (1.69 g, 5
mmol), 3,5-dihydroxy benzyl alcohol (0.28 g, 2 mmol), 1.38 g
K2CO3 and DMF (10 mL) were obtained 1.34 g (90%) as a
white solid.
EXPERIMENTAL
Materials
N-vinyl caprolactam (NVCL, 98%, Sigma) was distilled under
reduced pressure to remove the inhibitor and restored at 4
8C. CuCl were purified by stirring in acetic acid, washed with
ethanol, and then died in vacuum. 5, 5, 7, 12, 12, 14-
1H NMR (500 MHz, CDCl3, d): 4.65 (d, J 5 5.7 Hz, 2H; CH2O),
5.00 (s, 4H, PhCH2O), 5.06 (s, 8H, PhCH2O), 6.54 (t, J 5 2.1
Hz, 1H; CH), 6.59 (t, J 5 2.4 Hz, 2H; CH), 6.63 (d, J 5 2.1 Hz,
2
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4H; CH) 6.70 (d, J 5 2.1 Hz, 4H; CH), 7.33–7.45 (m, 20H,
Ar H).
Synthesis of G3-OH
G3-OH was synthesized by the same general procedure
described for the synthesis of G1-OH. From G2-Cl (3.81 g, 5
mmol), 3, 5-dihydroxy benzyl alcohol (0.28 g, 2 mmol),
K2CO3 (1.5 g), and DMF (10 mL) were obtained 3.3 g (81%)
as a white solid.
SCHEME 1 Synthesis of dendritic poly(benzyl ether)-b-poly(N-
vinylcaprolactam) block copolymers (Gn-b-PNVCL).
1H NMR (500 MHz, CDCl3, d): 4.57 (d, J 5 6.0 Hz, 2H; CH2O),
4.95 (s, 8H, PhCH2O), 5.01 (s, 16H, PhCH2O), 6.52 (m, 3H,
CH), 6.55 (d, J 5 1.8 Hz, 2H; CH), 6.64 (d, J 5 2.1 Hz, 4H; CH),
6.66 (d, J 5 2.1 Hz, 4H; CH), 7.27–7.40 (m, 40H, Ar H).
1H NMR (500 MHz, CDCl3, d): 4.47(s, 2H, CH2Cl), 4.95 (s, 8H,
PhCH2O), 5.01 (s, 16H, PhCH2O), 6.52 (m, 3H, CH), 6.55 (d,
J 5 1.8 Hz, 2H; CH), 6.64 (d, J 5 2.1 Hz, 4H; CH), 6.66 (d,
J 5 2.1 Hz, 4H; CH), 7.27–7.40 (m, 40H, Ar H).
Synthesis of G4-OH
G4-OH was synthesized by the same general procedure
described for the synthesis of G1-OH. From G3-Cl (0.41 g,
0.25 mmol), 3,5-dihydroxy benzyl alcohol (0.41 g, 0.25
mmol), K2CO3 (1.5 g), and DMF (10 mL) were obtained
0.42 g (76.5%) as a white solid.
Synthesis of G4-Cl
G4-Cl was synthesized by the same general procedure
described for the synthesis of G1-Cl. From G4-OH (0.70 g, 0.2
mmol), SOCl2 (0.10 mL, 1.4 mmol), NVP (1 mL) were
obtained 0.54 g (78%) as a white solid.
1H NMR (500 MHz, CDCl3, d): 4.60 (d, J 5 5.7 Hz, 2H; CH2O),
4.98 (s, 28H, PhCH2O), 5.04 (s, 32H, PhCH2O), 6.55–6.69 (m,
42H, CH), and 7.33–7.43 (m, 80H, Ar H).
1H NMR (500 MHz, CDCl3, d): 4.41 (s, 2H, CH2Cl), 4.98 (s,
28H, PhCH2O), 5.04 (s, 32H, PhCH2O), 6.55–6.69 (m, 42H,
CH), and 7.33–7.43 (m, 80H, Ar H).
Synthesis of Dendritic Benzyl Chlorides (Gn-Cl)
Synthesis of G1-Cl
Synthesis of Gn-b-PNVCL
To a solution of SOCl2 (0.5 mL, 6.92 mmol) in NMP (2mL)
was added the appropriate dendritic benzyl alcohol (1.00
equiv) at 0 8C under nitrogen, and stirred for 10 min at that
temperature. K2CO3 (6.96 g, 50.35 mmol) was added and
stirred for 1 h at room temperature. Then, DI water was
added to dissolve K2CO3. The resulting solution was
extracted with CH2CI2 (33); the combined extracts were
dried over MgSO4. After the solvent had evaporated, the
remaining product was purified by silica gel column chroma-
tography (petroleum ether/EtOAc, v/v 5 1.5:1) to yield a
white solid. The yield was 95% (2.03 g).
The Gn-b-PNVCL was synthesized utilizing ATRP techniques
as illustrated in Scheme 1. Typically,
a Schlenk flask
equipped with a magnetic stirrer was charged with NVCL
(1.39g, 12.5 mmol), CuCl (0.01 g, 0.085 mmol), Me6Cyclam
(0.03 g, 0.1 mmol), a mixture of 1, 4-dioxane and isopropa-
nol (300 lL), and pump-filled with nitrogen three times.
Then the initiator G1-Cl (0.03 g, 0.1 mmol) was added to the
flask under nitrogen. The reaction mixture was stirred for
4 h. The DI water was added and stirred for 24 h. The solu-
tion was filtered off and then transferred to a dialysis bag
(molecular weight cutoff 5 3500) and dialyzed against deion-
ized water for 7 days. The final product was dried in a vac-
uum oven, yielding a white solid.
1H NMR (500 MHz, CDCl3, d): 4.54 (s, 2H, CH2Cl), 5.06 (s,
4H, PhCH2O), 6.60 (t, J 5 2.1 Hz, 1H; CH), 6.67 (d, J 5 2.1 Hz,
2H; CH), 7.28–7.41 (m, 10H, Ar H).
Preparation of Micelles
Synthesis of G2-Cl
In a typical procedure, a copolymer (10 mg) was dissolved
in THF (3 mL). Then doubly distilled water (3 mL) was
added dropwise. The solution was dialyzed against distilled
water for 24 h to remove the THF. The obtained solution
was transferred into a 10 mL volumetric flask, followed by
dilution to the calibration mark with doubly distilled water
to obtain the micellar solution of 1.0 mg mL21. The obtained
micellar solution was kept for a week at room temperature,
and then was filtered through a 0.45 mm-pore membrane fil-
ter before both the mean size and morphology of nanopar-
ticles were determined by DLS and TEM, respectively. For
TEM studies, a drop of micellar solution was placed onto a
copper/carbon grid and the excess solution was blotted up
using a strip of filter paper, and then the sample was
allowed to dry at room temperature before observation.
G2-Cl was synthesized by the same general procedure
described for the synthesis of G1-Cl. From G2-OH (2.34 g,
3.15 mmol), SOCl2 (0.25 mL, 3.5 mmol), NVP (2 mL) were
obtained 2.1 g (90%) as a white solid.
1H NMR (500 MHz, CDCl3, d): 4.50 (s, 2H, CH2Cl), 5.96 (s,
4H, PhCH2O), 5.03 (s, 8H, PhCH2O), 6.53 (t, J 5 2.1 Hz, 1H;
CH), 6.57 (t, J 5 2.4 Hz, 2H; CH), 6.62 (d, J 5 2.1 Hz, 4H; CH)
6.67 (d, J 5 2.1 Hz, 4H; CH), 7.33–7.45 (m, 20H, Ar H).
Synthesis of G3-Cl
G3-Cl was synthesized by the same general procedure
described for the synthesis of G1-Cl. From G3-OH (1.25 g,
0.78 mmol), SOCl2 (0.13 mL, 1.75 mmol), NVP (2 mL) were
obtained 1.07 g (85%) as a white solid.
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growth medium was removed and replaced with the medium
containing three dilute solutions of G3-b-PNVCL (800
lL mL21) or G4-b-PNVCL (160 or 800 lg mL21), DMEM as
the negative control. Then, the cells were fixed using 2%
paraformaldehyde, blocked in 10% fetal calf serum at room
temperature for 20 min, incubated with FITC-labeled phalloi-
din (1 mg mL21) for 1 h. When fixed, the cells were washed
three times with PBS. The samples were imaged by fluores-
cence microscopy (Nikon TE2000).
RESULTS AND DISCUSSION
Synthesis and Characterization of Dendritic Poly(benzyl
ether)-b-poly(N-vinylcaprolactam) Block Copolymers
Four generations of dendritic poly(benzyl ether)-b-poly(N-
vinylcaprolactam) block copolymers (Gn-b-PNVCL) were syn-
thesized. Scheme 1 shows the synthetic route used for the
preparation of the Gn-b-PNVCL by ATRP of NVCL using four
generations of poly(benzyl ether) dendrons having benzyl
chloride focal points Gn-Cl as initiators and CuCl/Me6Cyclam
as catalyst. 1H NMR, FTIR, and GPC analyses were then
used to confirm the formation of Gn-b-PNVCL copolymers.
Figures 1(a) and 2(a) as well as Supporting Information Fig-
ures S1a and S2a show the 1H NMR spectra of Gn-b-PNVCL
in CDCl3. All characteristic resonances in dendritic poly(ben-
zyl ether) blocks and PNVCL blocks can be found. The peaks
at 6.55–6.58, 6.66–6.68, and 7.38 ppm [Fig. 1(a)] are attrib-
uted to the protons of dendritic poly(benzyl ether), whereas
the peaks at 1.46–1.81, 3.23 and 4.41–4.51 ppm are attrib-
uted to the protons of PNVCL block. The Mn,NMR values of
Gn-b-PNVCL were calculated based on the integration ratio of
the signal at 6.55–6.58 ppm corresponding to protons e and
f in the dendritic poly(benzyl ether) to that at 3.23 ppm cor-
responding to the methylene protons j of the PNVCL ring in
the a-position to the N atom of the LDBCs. As showed in
Table 1, the Mn,NMR values of the Gn-b-PNVCL are close to
their Mn,theo values.
FIGURE 1 1H NMR spectra of G1-b-PNVCL in CDCl3 (a) and D2O (b).
For 1HNMR spectra studies, the micellar solution was pre-
pared as above-mentioned and then freeze-dried for 48 h.
The lyophilized micelles were dispersed in D2O.
The CMC of the copolymers solution was determined with a
fluorescence spectrometer (Hitachi F-4500 luminescence
spectrophotometer) using pyrene as a hydrophobic probe.42
Temperature-Dependent Turbidimetry
The LCST of the aqueous solution of the polymer was mea-
sured at 500 nm with a UV–vis spectrometer (CARY UV-50,
VARIAN) equipped with a water-circulation heating stage.
The polymers were dissolved in deionized water at different
concentrations (0.04–0.6 mg mL21). The heating rate was
0.2 8C min21. The LCST value was defined as the tempera-
tures corresponding to 1% decreases of transmittance.56
In Vitro Cytotoxicity Testing
In vitro cytotoxicity of G3-b-PNVCL and G4-b-PNVCL was esti-
mated by MTT viability assay against mouse L929 fibro-
blasts. L929 cells were seeded in 96 well plates at 4 3 104
cells mL21 in 90 lL of Dulbecco’s modified Eagle’s medium
(DMEM; Sigma), containing 10% fetal bovine serum, and
incubated at 37 8C in a 5% CO2 atmosphere for 24 h, fol-
lowed by adding block copolymer solution (10 lL in normal
saline) at different concentrations. The cells were grown for
FTIR analysis (Supporting Information Fig. S3) of Gn-b-
PNVCL reveals several characteristic peaks that can be corre-
lated with the expected molecular structure of the LDBCs.
The FTIR spectrum of G1-b-PNVCL is shown in Supporting
another 48 h. Then, 10 lL of MTT solution (5 mg mL21
;
Sigma) was added to each well. After incubating the cells for
4 h, the medium containing unreacted MTT was carefully
removed. The obtained blue formazan crystals were dis-
solved in 100 lL per well of dissolving solution (10%
sodium dodecylsulfate, 5% isobutanol, 0.012 mol L21 HCl)
and incubated overnight at 37 8C. Negative (normal saline)
control wells were treated as above-mentioned. The absor-
bance at 570 and 630 nm was detected using an ELISA
reader (Bio-Rad, model 680). Cell viability (as a percentage
of the negative control) was calculated from the optical den-
sity values.
Immunofluorescence Microscopy
For F-actin staining, L929 cells were seeded onto glass cov-
erslips in a 24 well plate at a density of 1 3 104 cells per
well in 1mL of complete medium for 24 h after which the
FIGURE 2 1H NMR spectra of G2-b-PNVCL in CDCl3 (a) and D2O (b).
4
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TABLE 1 Characterizations of Gn-b-PNVCL
PNVCL/dendrond
(wt %)
a
b
Mn,th
Mn,GPC
Mn,NMR
(g mol21
Conversion
(%)
Sample
(g mol21
)
(g mol21
)
)
PDI 5 Mw/Mn
DPc
G1-b-PNVCL
G2-b-PNVCL
G3-b-PNVCL
G4-b-PNVCL
10064.5
9398.5
9969.5
11667.5
7757
10,126
10,578
11,457
12,853
1.02
1.21
1.25
1.24
70
62
60
60
71
71
71
69
97/3
21,316
22,194
23,326
93/7
87/13
75/25
a
c
The theoretic molecular weight was calculated by the formula:
The degree of polymerization of PNVCL block was calculated using
Mn, th 5 Mmonomer 3 ([monomer]/[Gn-Cl]) 3 Conversion% 1 Minitiation
Mn and PDI were determined by GPC.
.
Mn,NMR
Dendritic poly(benzyl ether).
.
b
d
Information Fig. S3a as a typical example. The FTIR spec-
trum of G1-b-PNVCL shows that the characteristic peaks at
1632 cm21 for stretching vibrations of acyl group in the
amide moieties of PNVCL, 1541 and 1475 cm21 for the phe-
nyl groups, 1197 cm21 for CAO stretching vibration of den-
dritic poly(benzyl ether) were observed.
CMC value that can be determined by intersecting the two
straight lines, varied within 0.0162–0.0013 mg mL21 for
these LDBCs. These CMC values are comparable to those
reported for some LDBCs based on flexible dendritic ali-
phatic polyesters which may afford additional peripheral
groups used for providing functionality to the LDBCs,57
which illustrates the effectiveness of the cone-shaped
arrangement of the LDBCs and the multi-branched poly(ben-
zyl ether) structure for improving the stability. Furthermore,
it should be noted that the CMC of Gn-b-PNVCL decreased
with increasing the generation of the dendritic poly(benzyl
ether). This result demonstrated that the generation of the
dendritic poly(benzyl ether) impacted the CMC of Gn-b-
PNVCL greatly, and the high generation dendritic poly(benzyl
ether) stabilized micellar aggregates due to its high hydro-
phobic composition.
Figure 3 gave the GPC traces of Gn-b-PNVCL. All copolymers
show unimodal GPC curves with narrow molecular weight
distributions (Mw/Mn ꢀ 1.25; Table 1), which indicated that
the polymerizations were controlled. However, the molecular
weights from gel permeation chromatography (GPC) were
not in good agreement to the targeted values as conventional
calibration using linear PS standards was used.
Self-Organization in Aqueous Media of Dendritic
Poly(benzyl ether)-b-poly(N-vinylcaprolactam) Block
Copolymers
The self-assembly of Gn-b-PNVCL forming micellar aggregates
1
The presence of a hydrophobic dendritic poly(benzyl ether)
and hydrophilic linear PNVCL chain means that these amphi-
philic LDBCs are expected to be able to self-assemble into
micelle-like nanoparticles in water. The critical micelle con-
centration (CMC) of amphiphilic copolymers was one of the
most important parameters of characterizing the thermody-
namic stability of nanoparticles in aqueous solution. Thus,
the CMC of Gn-b-PNVCL was determined by a fluorescence
technique with pyrene as a probe. The ratios of the intensity
at 383–374 nm from the emission spectra of pyrene were
plotted against the concentration of Gn-b-PNVCL. As showed
in Figure 4, the I383/I374 ratios remain fairly constant below
a certain concentration and then increase remarkably above
that concentration, indicating the formation of micelles. The
in aqueous was verified by H NMR spectra. In CDCl3, where
1
micellar formation was not expected, all H NMR resonances
attributed to poly(benzyl ether) and PNVCl were detected as
shown in Figures 1(a) and 2(a) and Supporting Information
1
Fig. S1a-S2a. When the H NMR spectra of Gn-b-PNVCL were
recorded in D2O, the benzyl proton peaks at 6.55–6.58, 6.66–
6.68, and 7.38 ppm disappeared [Figs. 2(b) and 3(b), Sup-
porting Information Fig. S1b-S2b], indicating that the mobil-
ity of dendritic poly(benzyl ether) segments has been
FIGURE 4 Plots of I383/I374 versus logarithm of concentration of
Gn-b-PNVCL. [Color figure can be viewed at wileyonlinelibrary.
com]
FIGURE 3 GPC traces of Gn-b-PNVCL.
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FIGURE 5 TEM images of the formed micelles by (a) G1-b-PNVCL, (b) G2-b-PNVCL, (c) G3-b-PNVCL, and (d) G4-b-PNVCL.
restricted by the formation of core-shell micellar structure in
the aqueous environment.
poly(benzyl ether) cores.60 For G4-b-PNVCL with a weight
fraction of 75 wt % PNVCL, spherical micelles with a mean
diameter of 80–150 nm as well as rod-like LCVs are obtained
[Fig. 5(d)]. The size and shape of the rod-like LCVs are simi-
lar to those of LCVs from G3-b-PNVCL. However, there are
some smaller vesicles and spherical micelles in the rod-like
LCVs, which is different from the LCVs from G3-b-PNVCL. The
above experimental phenomenon shows that the self-
assembly behavior of Gn-b-PNVCL copolymers can be affected
by the generation of the dendritic poly(benzyl ethers) block.
Gn-b-PNVCL is first dissolved in THF, and micellization is
induced by the dropwise addition of water, followed by dial-
ysis, and the resulting dialyzed aqueous solution was sub-
jected to an investigation of their self-assembled morphology
via TEM studies. As shown in Figure 5, the multiple mor-
phologies of the aggregates made from the LDBCs of differ-
ent generations. For G1-b-PNVCL, when the weight fraction
of hydrophilic PNVCL block was 97 wt % within the LDBC,
irregularly spherical micelles with a mean diameter of 50–
130 nm were observed in Figure 5(a). However, when the
weight fraction of PNVCL block decreased to 93 wt % within
G2-b-PNVCL, the vesicles with a mean diameter of 130–
168 nm and a wall thickness of 30 nm were obtained [Fig.
5(b)]. The vesicles are dispersed very well and almost no
cohesion happens during drying, which may be ascribed to
Thermoresponsive Properties of Dendritic Poly(benzyl
ether)-b-poly(N-vinylcaprolactam) Block Copolymers
UV–vis spectrophotometer was first used to study the trans-
mittances of Gn-b-PNVCL aqueous solution as a function of
temperature with various concentrations. As shown in Figure
6, all the copolymers were transparent and colorless at the
temperature below LCST (the transmittance of the solutions
is close to 100%) and start to aggregate (became opaque) at
LCST, which leads to a significant transition in the transmit-
tance. The LCSTs of the copolymers Gn-b-PNVCL decreased in
the order of G1-b-PNVCL > G2-b-PNVCL > G3-b-PNVCL > G4-b-
PNVCL at all concentrations tested (Supporting Information
Fig. S4), demonstrated that the generation of the LDBCs
impacted the phase transition greatly, and the high genera-
tion LDBCs facilitated the collapse of the copolymers. It is
well known that the LCST of thermoresponsive polymers can
be adjusted by the composition of the hydrophobic and
hydrophilic units. Generally, increasing hydrophobic seg-
ments leads to a decrease in the LCST of the polymer in
aqueous solution and hydrophilic units had the tendency to
increase LCST.61 In this case, Gn-b-PNVCL copolymers pos-
sess different hydrophilic and hydrophobic compositions.
The numbers of hydrophobic phenyl groups increased signif-
icantly with the generation. The high generation LDBCs
ꢀ
the P-P aromatic stacking of the Frechet-type dendrons
increase the stability of the bilayer vesicle structures.58 For
G3-b-PNVCL with a weight fraction of 87 wt % PNVCL, under
the same self-assembly conditions, unique rod-like large
compound vesicles (LCVs) are the dominant type of aggre-
gates; 4–10 vesicles with a mean diameter of 270–560 nm
aggregated together to form rod-like LCVs with tubularly
packed hoops [Fig. 5(c)]. These rod-like LCVs have relatively
uniform diameter (ca. 560 nm), and their lengths are in the
range of 1.6–2.3 mm. Spherical or hemispherical caps are
clearly seen at the rod ends. The rod-like LCVs may be
ascribed to the association of individual vesicles like the
intermicellar aggregation.59 G3-b-PNVCL first self-assembled
into vesicles and then the vesicles further aggregated into
rod-like LCVs, which most likely the length of the PNVCL
block is too short to outweigh the hydrophobic-hydrophobic
or van der Waals interactions between the exposed dendritic
6
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FIGURE 6 Transmittance measurements as a function of temperature for different concentrations of G1-b-PNVCL (a), G2-b-PNVCL
(b), G3-b-PNVCL (c), and G4-b-PNVCL (d). [Color figure can be viewed at wileyonlinelibrary.com]
possessed more phenyl groups, which would enhance the
hydrophobicity of the linear-dendritic copolymers and thus
resulted in lower LCSTs. The concentration dependence of
the LCSTs of these linear-dendritic copolymers was also
studied. Figure 6 shows the transmittance of Gn-b-PNVCL as
a function of temperature at different concentrations. The
LCST values of Gn-b-PNVCL have strong concentration depen-
dence. Their LCST values increased with decreasing the
linear-dendritic copolymer concentration, and the lower the
polymer concentration were, the broader the temperature
range exhibited by the decrease in transmittance. Such a
phenomenon is known to be a general trend in the concen-
tration effect on the LCST of PNVCL-containing block
copolymers.38
The thermoresponsive phase transition behavior of Gn-b-
PNVCL copolymers was further investigated by dynamic light
scattering (DLS). Figure 7 shows the temperature depen-
dence of the average hydrodynamic radii (Rh) of the LDBCs
in aqueous solution at a concentration of 1.0 mg mL21 upon
heating. The LCST values are in good agreement with those
obtained from the turbidimetric studies and demonstrate
that the LCSTs of the LDBCs depend on the generation of the
dendritic poly(benzyl ethers) and decrease from 36 to 33 8C
when the generation is increased from the first to fourth,
62
ꢀ ^
Jerome and coworkers
noted that the transmission of
poly(ethylene glycol)-b-poly(N-vinylcaprolactam) (PEG-b-
PNVCL) block copolymers with shorter PNVCL block length
cannot decrease to zero.
A similar phenomenon was
observed here for our linear-dendritic copolymers (Fig. 6).
The remaining transmittance at high temperature is rather
low (few percent) when the concentration of the copolymer
was more than 0.2 mg mL21 for G1-b-PNVCL, 0.08 mg mL21
for G2-b-PNVCL, G3-b-PNVCL, and G4-b-PNVCL. The residual
transmittance is considerable throughout (15–35%) as the
concentration and generation of the linear-dendritic copoly-
mers decrease. This phenomenon was probably attributed to
the relative diminished content of thermoresponsive PNVCL
segment in G2-b-PNVCL, G3-b-PNVCL, and G4-b-PNVCL.
FIGURE 7 Plots of hydrodynamic radii (Rh) of the micelles
formed by Gn-b-PNVCL in aqueous solution as a function of
temperature from DLS measurements. [Color figure can be
viewed at wileyonlinelibrary.com]
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TABLE 2 Cell Viability Values after 48 h of Incubation with G3-
b-PNVCL and G4-b-PNVCL at 37 8C
adherent cells growing as a confluent monolayer [Fig. 8(a)].
Figure 8(b–d) reflect the morphology of cells after they were
cultured in a solution of G3-b-PNVCL (800 lg mL21) or G4-b-
PNVCL (160 or 800 lg mL21) for 48 h. The dimension,
shape, and density of the cells in the solution of the polymer
were very similar to those in the negative control [Fig. 8(a)],
indicating that no or little damage could be detected. These
results demonstrate that the obtained LDBCs are potentially
applicable in biomedical fields.
Concentration (lg/mL)
1.3
6.4
32
160
800
Viability (%, G3-b-PNVCL) 96.1 101.7 94.4 96.7
Viability (%, G4-b-PNVCL) 91.6 91.1
115.0
99.2 104.5 97.5
due to the increased the numbers of hydrophobic phenyl
groups in the LDBCs.
In Vitro Cytotoxicity of Dendritic Poly(benzyl ether)-b-
poly(N-vinylcaprolactam) Block Copolymers
CONCLUSIONS
A
series of new well-defined linear-dendrimeric block
To evaluate the biocompatibility of the obtained LDBCs, the
in vitro cytotoxicity was measured in mouse L929 fibroblasts
using the MTT assay, a valid and reliable measure of viability
based on the succinic dehydrogenase activity of cultured
cells. The results from the dose-response relationship for the
third or fourth generation of the copolymer G3-b-PNVCL or
G4-b-PNVCL at concentrations ranging from 1.3 to 800
lg mL21 are shown in Table 2. It was demonstrated that
cells remained viable even when the concentration of the
LDBCs were up to 800 lg mL21, suggesting their good bio-
compatibility to L929 cells. The cell viabilities were >95%
for G3-b-PNVCL and >90% for G4-b-PNVCL at the concentra-
tions studied.
copolymers (LDBCs) have been synthesized in which the lin-
ear block is PNVCL and the dendrimeric block is poly(benzyl
ether) of generation G1–G4. Their molecular structures and
solution properties were characterized in detail by 1H NMR,
FTIR, GPC, fluorescent spectroscopy, TEM, turbidity, and DLS
measurements. A morphological transition of the polymeric
aggregates in water from irregularly spherical micelles,
vesicles to rod-like LCVs and eventually to the coexistence of
spherical micelles and rod-like LCVs has been demonstrated
by increasing the generation number of the hydrophobic
dendritic block. The LDBCs exhibit thermally induced phase
transition in aqueous solution and their LCST decrease with
increase in the generation of the dendritic poly(benzyl
ethers) and the concentrations of the LDBCs solutions. The
biocompatibility of the LDBCs is evaluated, and they are
proven to be nontoxic to L929 cells.
Furthermore, the F-actin staining results also demonstrate
the good biocompatibility of G3-b-PNVCL and G4-b-PNVCL.
Usually, L929 mouse fibroblasts are large, spindle-shaped,
FIGURE 8 Fluorescence microscope micrographs of mouse L929 fibroblasts grown on DMEM (a), on G3-b-PNVCL [b (800
lg mL21)], and G4-b-PNVCL [c (160 lg mL21), d (800 lg mL21)] expressing F-action. [Color figure can be viewed at wileyonlineli-
brary.com]
8
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge support for this work of
the National Natural Science Foundation of China (21264017
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