A thermoresponsive amphiphilic dendron - Synthesis, characterization, and self-assembled micelles for controlled drug release

Article abstract of DOI:10.1139/v2012-038

A new third-generation thermoresponsive amphiphilic dendron consisting of a hydrophobic poly(benzyl ether) dendritic core and hydrophilic oligo(ethylene glycol) peripheries was synthesized by an efficient convergent approach. Its structure was confirmed by ¹H NMR, ¹³C NMR, IR, GPC, MALDI-TOF MS, and elemental analysis. Turbidity and dynamic light scattering (DLS) measurements demonstrated that the dendron showed a reversible temperature-dependent phase-transition behavior in aqueous solution and its lower critical solution temperature (LCST) was lower than that of the corresponding second-generation dendron, indicating the dependence of LCSTs on the generation of dendrons. Fluorescent spectroscopy and TEM studies revealed that the dendron would self-assemble into nanospherical micelles with a very low critical micelle concentration (CMC) in water. The core-shell structure of the micelles was proved by ¹H NMR analyses of the micelles in D ₂O. The drug-loading capacity of the dendron micelles is about 29 wt % for podophyllotoxin (POD) used as a model drug, and in vitro release tests showed a desired thermoresponsive drug-release behavior. These results indicate that the dendron is promising as stimuli-responsive material for biomedical applications.

Full text of DOI:10.1139/v2012-038

600
A thermoresponsive amphiphilic dendron —
Synthesis, characterization, and self-assembled
micelles for controlled drug release
Li Xu, Lidong Shao, Minqi Hu, Lin Chen, and Yunmei Bi
Abstract: A new third-generation thermoresponsive amphiphilic dendron consisting of a hydrophobic poly(benzyl ether)
dendritic core and hydrophilic oligo(ethylene glycol) peripheries was synthesized by an efficient convergent approach. Its
1
structure was confirmed by H NMR, 13C NMR, IR, GPC, MALDI-TOF MS, and elemental analysis. Turbidity and dy-
namic light scattering (DLS) measurements demonstrated that the dendron showed a reversible temperature-dependent
phase-transition behavior in aqueous solution and its lower critical solution temperature (LCST) was lower than that of the
corresponding second-generation dendron, indicating the dependence of LCSTs on the generation of dendrons. Fluorescent
spectroscopy and TEM studies revealed that the dendron would self-assemble into nanospherical micelles with a very low
1
critical micelle concentration (CMC) in water. The core-shell structure of the micelles was proved by H NMR analyses of
the micelles in D2O. The drug-loading capacity of the dendron micelles is about 29 wt % for podophyllotoxin (POD) used
as a model drug, and in vitro release tests showed a desired thermoresponsive drug-release behavior. These results indicate
that the dendron is promising as stimuli-responsive material for biomedical applications.
Key words: amphiphilic dendron, poly(benzyl ether) dendritic core, oligo(ethylene glycol) peripheries, thermoresponsive mi-
celles, controlled drug release.
Résumé : Utilisant une approche impliquant une synthèse efficace et convergente, on a réalisé la synthèse d’un nouveau
dendron amphiphile thermosensible de troisième génération formé d’un coeur dendritique, un poly(éther benzylique) hydro-
phobe, et des périphéries hydrophiles à base d’oligo(éthylèneglycol). On en a confirmé la structure résonance magnétique
1
nucléaire du H et du 13C, infrarouge, chromatographie en phase gazeuse, spectrométrie de masse en temps de vol avec ioni-
sation par désorption au laser d’une matrice (« MALDI-TOF MS ») et par analyse élémentaire. Des mesures de turbidité et
de diffusion de la lumière dynamique (DLD) ont permis de démontrer que, en solution aqueuse, le dendron donne lieu à un
comportement impliquant une transition de phase réversible en fonction de la température et que sa température de solution
critique la plus basse (TSCB) est plus basse que celle du dendron correspondant de deuxième génération, ce qui met en évi-
dence que la température de solution critique la plus basse (TSCB) varie en fonction de la génération des dendrons. Des étu-
des de spectroscopie de fluorescence et de microscopie électronique de transmission (MET) mettent en évidence le fait que,
dans l’eau, le dendron pourrait donner un auto-assemblage en micelles nanosphèriques avec une concentration micellaire cri-
1
tique (CMC) très basse. Faisant appel à des analyses de RMN du H, on a pu déterminer que, dans le D2O, la structure des
micelles correspond à coeur en forme de coquille. La capacité de charge de médicament par les micelles de dendron est
d’environ 29 % en poids pour la podophyllotoxine (POD) utilisée comme médicament modèle et des essais d’émission in vi-
tro ont permis de démontrer un comportement recherché d’émission thermosensible des médicaments. Ces résultats indi-
quent que le dendron est un matériel stimuli-sensible prometteur pour des applications biomédicales.
Mots‐clés : dendron amphiphile, poly(éther benzylique) à coeur dendritique, oligo(éthylèneglycol), périphéries, micelles ther-
mosensibles, émission contrôlée de médicament.
[Traduit par la Rédaction]
effect9 for applications such as biomedicine and catalysis.10–13
Introduction
Thermoresponsive dendrimers can be constructed by attach-
ing thermoresponsive groups or polymers to the periphery or
the core of the dendrimer. Dendrimers with properly bal-
anced hydrophilic and hydrophobic units can also act as tem-
perature-sensitive dendrimers.14 Poly(N-isopropylacrylamide)
(PNIPAM) is a representative polymer exhibiting thermores-
ponsive behavior. It shows a dissolution–precipitation transition
In recent years, a growing interest has been evident in the
development of dendrimers that are responsive to external
stimuli, such as pH,1,2 temperature,3,4 light,5,6 and redox
state.7,8 One of the most appealing stimuli-responsive species
is the thermoresponsive dendrimer, since such a system com-
bines the advantages of temperature sensitivity and dendritic
Received 21 February 2012. Accepted 8 May 2012. Published at www.nrcresearchpress.com/cjc on 4 July 2012.
L. Xu, L. Shao, M. Hu, L. Chen, and Y. Bi. Yunnan Normal University, College of Chemistry and Chemical Engineering, Yunnan
Normal University, Kunming 650092, P.R. China.
Corresponding author: Yunmei Bi (e-mail: yunmeibi@hotmail.com).
Can. J. Chem. 90: 600–607 (2012)
doi:10.1139/V2012-038
Published by NRC Research Press

Xu et al.
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in aqueous solution at 32 °C. N-Isopropylacrylamide (NIPAM)
has been incorporated into linear and dendritic macromole-
cules to obtain thermoresponsive copolymers and den-
drimers.15–20 For example, Kimura et al.21 prepared the first
thermoresponsive dendrimer by polymerization of NIPAM
from the termini of a poly(propyleneimine) dendrimer with
terminal thiol groups. The core of the dendrimer was a co-
balt complex, which catalyzed oxidation of the thiol com-
pounds.
the second-generation dendron alcohol G2b. Chlorination of
G2b with SOCl2 afforded the corresponding chloride G2c.
In this study, each dendron was purified simply by solvent
extraction or dialysis to remove the earlier generation den-
dron. Compared with the synthetic strategy of the corre-
sponding first-generation dendron, the reaction conditions to
synthesize G2b were modified by adjusting the molar ratio
of LAH to G2a from 2:1 to 3:1 and prolonging the reaction
time from 16 to 20 h. The reaction time of G2b with SOCl2
was also prolonged from 3 to 4 h. As a result, the purifica-
tion of G2b and G2c required only solvent extraction. The
alkylation of methyl gallate with G2c by using K2CO3 as the
base in N, N-dimethylformamide (DMF) provided analyti-
cally pure third-generation dendron G3, requiring only a dial-
ysis workup. None of the dendrons required column
chromatography for analytically pure material, thus facilitat-
ing scale-up. All new compounds were obtained as oily
Polyethylene glycol (PEG), an uncharged, nontoxic, and
nonimmunogenic water-soluble polymer, has found diverse
applications in biological sciences, personal care products,
and industrial additives.22 However, PEG possesses a lower
critical solution temperature (LCST) above 100 °C, which is
too high and may be a limitation to its widespread use in the
biomedical field.23 Recently, PEGylated polymers have been
explored by incorporating PEG or oligo(ethylene glycol)
(OEG) units into other macromolecules. They show lower
LCSTs and are promising alternatives to PNIPAM for appli-
cations in drug delivery systems.24–28 PEG is also useful for
the preparation of temperature-sensitive dendrimers. For ex-
ample, Li et al.29 synthesized dendrimers containing temper-
ature-sensitive oligoethylene glycol as a building block.
These unique dendrimers showed fast and sharp transitions
around their LCSTs. Their thermoresponsive behaviors are
dependent on both the generation and the terminal structure.
Some amphiphilic PEGylated dendrons that are parts of
dendrimers have temperature-dependent phase-transition be-
havior. Moreover, these dendrons, composed of hydrophobic
and hydrophilic parts, can self-assemble into dendritic mi-
celles. For instance, Asthmanikandan et al.9 reported on ther-
mosensitive biaryl-based dendrons with a pentaethylene
glycol monomethyl ether and a decyl chain. These amphi-
philic dendrons exhibited generation-dependent temperature
sensitivity and were capable of forming micelles and reverse
micelles in polar and apolar solvents, respectively. Chang and
Dai30 synthesized dendrons consisting of hydrophobic oligo
(p-phenylene vinylene) core branches and hydrophilic oligo
(ethylene oxide) terminal chains. These dendrons showed a
LCST and the critical micellization behavior. The unusual
properties of these amphiphilic PEGylated dendrons are at-
tractive for potential application in controlled drug delivery
systems.
1
liquids and their structures were characterized by H NMR
and 13C NMR spectra (Supplementary data Figs. S1–S3;
Fig. 1), FT-IR, as well as ESI-MS or MALDI-TOF MS. G3
was further characterized by GPC and elemental analysis.
GPC analysis of G3 showed a single peak (PDI = 1.05,
Fig. 2). The Mn measured by GPC for G3 was much lower
than the calculated one using calibration with linear polysty-
rene. This is attributed to the difference between the hydro-
dynamic volumes of polystyrene and G3 and is consistent
with the studies of polyether dendrimers by Fréchet and co-
workers.32 However, MALDI-TOF MS of G3 showed a sin-
gle signal at m/z ([M + Na]+) = 4624 (calcd mass, 4599),
indicating the formation of G3.
Formation of micelles
The presence of a hydrophobic poly(benzyl ether) branch
unit and hydrophilic oligo(ethyleneoxy) periphery means that
these amphiphilic dendrons can potentially be precursors to
nanoparticles with an oligo(ethyleneoxy) corona and a poly
(benzyl ether) core upon dissolution in water, which is a se-
lective solvent. The formation of the hydrophobic poly(ben-
zyl ether) domain can be confirmed by 1H NMR spectra
with CDCl3 and D2O as locking solvents for G3. In CDCl3,
1
where micellar formation was not expected, all H NMR res-
onances attributed to poly(benzyl ether) and oligo(ethyl-
eneoxy) units were detected as shown in Fig. 1a. However,
1
the H NMR spectrum in D2O showed an intensity reduction
Usually, the purification of synthesized dendrons or den-
drimers requires column chromatography, which not only is
tedious to perform but also unavoidably leads to losses of
product. In this paper, we described the column-free synthe-
sis, and thermoresponsive and micellar behaviors of a third-
generation amphiphilic dendron containing a hydrophobic
poly(benzyl ether) dendritic core and 27 hydrophilic diethyl-
enoxy methyl ethers at its periphery. The controlled release
of the model drug podophyllotoxin (POD) using the thermor-
esponsive micelles was also studied in detail.
of poly(benzyl ether) resonances resulting from the sup-
pressed molecular motion of the aggregated hydrophobic
branch units (Fig. 1b). The small broad signals in the NMR
spectrum indicate restricted motions of these protons within
the core of nanoparticles. This result is evidence for a core-
shell type formation with a hydrophobic inner core and a
1
hydrophilic shell. Similar trends in H NMR spectra are con-
sistent with amphiphilic block copolymer systems.33,34
The formation of micelles from the third-generation den-
dron G3 was also examined by the detection of critical mi-
celle concentration (CMC) relying on
a fluorescence
Results and discussion
technique with pyrene as the probe because pyrene preferen-
tially partitions into the hydrophobic core of micelles from
water.35 The intensity ratio of I341/I337 vs the logarithm of
concentrations of G3 in the pyrene excitation spectra was
plotted in Fig. 3a. It is seen that the I341/I337 ratios remain
fairly constant below a certain concentration and then increase
Synthesis and characterizations of dendrons
The synthetic procedures of the dendrons are summarized
in Scheme 1. The second-generation dendron G2a was syn-
thesized according to our previously reported method.31 G2a
was reduced with lithium aluminum hydride (LAH) to give
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Can. J. Chem. Vol. 90, 2012
Scheme 1. Synthesis of dendron G3. Reagents and conditions: (a) LiAlH4 (LAH), tetrahydrofuran (THF), room temperature (rt), 20 h; (b)
SOCl2, N, N-dimethylformamide (DMF), dichloromethane (DCM), rt, 3 h; (c) K2CO3, DMF, 70 °C, 24 h.
remarkably above that concentration, indicating the formation
of micelles. From this plot, a CMC of 7.7 × 10–6 mol/L was
obtained from the intersection of two straight lines: the
baseline and the rapidly rising I341/I337 line (Fig. 3b), which
is lower than the one of the second-generation dendron G2a
(2.2 × 10–5 mol/L).31 This could be attributed to the fact
that G3 is larger than G2a, which facilitates the aggregation
of the dendron. These CMC values are comparable to those
reported for some other amphiphilic dendrons and linear co-
polymers.9,36 The low CMC values mean that the dendrons
can self-assemble to form nanoscaled core-shell micelles at
a lower compound concentration, which is suitable for drug
delivery in very dilute aqueous milieux such as body
fluids.37
Micelle morphology was investigated by transmission elec-
tron microscopy (TEM). It can be seen from Fig. 4 that den-
dron G3 forms spherical micelles with a core-shell (corona)
structure and an average diameter of about 60 nm. The mi-
celles made from G3 were bigger than those from G2a31 be-
cause of its larger core and peripheries.
ture. The concentration dependence of the LCST was also
investigated in the range of 0.1–2.1 mg/mL and the results
are plotted in Fig. 5. For comparison, the respective curve of
dendron G2a31 was also added (Fig. 5b). Dendron G3 dis-
played an obvious phase transition in water and its LCST val-
ues decreased with increasing concentration from 0.3 to
1.5 mg/mL. Compared with dendron G2a, G3 shows lower
LCSTs at all concentrations tested, demonstrating the de-
pendence of LCSTs on the generation of dendrons, which
was in correspondence with the report by Aathimanikandan
et al.9 The reason may arise from the increased size of the
dendrons with the increasing generation; thus, the interaction
of the peripheral oligo(ethyleneoxy) groups would take place
more efficiently with the increasing generation of the den-
dron, which leads to the dehydration of the terminal oligo
(ethyleneoxy) groups at a lower temperature with increasing
generation.3
The thermoresponsive behavior of dendrons G2a and G3
was also analyzed by dynamic light scattering (DLS). The
hydrodynamic diameter (Dh) of the dendrons are apparently
influenced by the temperature, as shown in Fig. 6. At temper-
atures lower than the LCSTs, the two dendrons are easily
solubilized in water so that the amphiphilic dendrons self-as-
semble into core-shell micelles with a low Dh of approxi-
mately 50–60 nm. However, a dramatic increase in Dh was
Thermoresponsive behavior
To investigate whether the third-generation dendron G3
exhibits a thermal response, we examined the optical trans-
mittance of a G3 aqueous solution as a function of tempera-
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Xu et al.
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1
Fig. 1. H NMR spectra of G3 in CDCl3 (a) and D2O (b).
Fig. 2. GPC trace of G3 with Mn (theory) = 4599 Da and
Mn (GPC) = 1666.
Controlled drug release
Hydrophobic drugs can be physically incorporated and sta-
bilized in the micellar hydrophobic inner core by hydropho-
bic interaction. Podophyllotoxin (POD), an anticancer drug
with poor solubility in water, was used as a model drug to
investigate the potential application of dendron G2a and G3
micelles in controlled drug release. The amount of drug in-
corporated into the micelles was measured by a UV spec-
trometer. The results showed that POD was successfully
incorporated into micelles of G2a and G3, and the drug-
loading content (DLC) and entrapment efficiency (EE) were
about 21.24% and 77.81% for G2a, and 28.71% and 94.95%
for G3, respectively, which are higher than those of the tradi-
tional micelles.37 Furthermore, the amount of POD intro-
duced into the micelle was dependent on the generation of
the dendrons. The DLCs and EEs of G3 with more hydro-
phobic phenyl groups are higher than those of G2a with
fewer hydrophobic phenyl groups. To obtain information
about the release of POD from G2a and G3 micelles, the
drug-loaded G2a and G3 micelles were dissolved in PBS
and dialyzed against PBS. Figure 7 shows the release profile
of POD from G2a and G3 micelles. In contrast with the
rapid release of the free drug from a cellulose membrane
(99% released in 4 h), the release from the dendritic micelles
was slow and showed sustained characteristics. The genera-
tion of dendrons also affects drug release. The amount of
drug released and the rate of release were greater for G3
than for G2a micelles. This could be ascribed to steric hin-
drance of crowded end functional groups in higher genera-
tions and weaker interaction between these functional groups
observed once the temperature increased to near the LCST
(58 and 54 °C, respectively), which indicated dendron aggre-
gation. Dendron G2a requires a higher temperature to induce
aggregation than dendron G3 does. The aggregation process
was completely reversible, and the aggregates became soluble
again below the onset temperature. These are consistent with
the results of turbidimetry measurement.
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Fig. 3. (a) Excitation spectra of pyrene in water at different concentrations of G3. (b) Plot of the intensity ratio I341/I337 vs the logarithm of the
concentration of G3.
Fig. 4. Transmission electron microscopy (TEM) image of G3 mi-
celles.
dendritic core and hydrophilic oligo(ethylene glycol) periph-
eries using a simple convergent approach. The dendron ex-
hibited a thermally induced phase transition in aqueous
solution and its LCST increased with decreasing concentra-
tion over the range of 0.3–2.1 mg/mL of the dendron in
aqueous solution. Studies of the association behavior of the
synthesized dendron in aqueous solution revealed that the
dendron is capable of forming spherical micelles of around
60 nm in diameter. Compared with its second-generation
counterpart, the dendron exhibited lower LCST and CMC.
The self-assembled dendron micelles exhibited high drug-
loading capacity and thermoresponsive drug release behavior.
These results indicate that the thermosensitive amphiphilic
dendron possesses promising potential applications in the bi-
omedical field.
and the drug molecules. A similar trend was observed in linear-
dendrimer block copolymers containing PEG studied by
Adeli et al.38 To evaluate the effect of the temperature stim-
ulus on release of POD from the thermoresponsive micelles,
the POD release from G3 micelles was examined in PBS
solution at 25, 37, and 55 °C. As shown in Fig. 8, the
POD release rate and amount increased with increasing
temperature under constant pH (7.4) . A slow release was
observed owing to the greater stability of micelles when
the temperature was below the LCST (20 and 37 °C). How-
ever, when the temperature was above the LCST (55 °C),
drug release was accelerated because of the temperature-
induced structure change of the micelles. At a temperature
above the LCST, the hydrophilicity of the micellar shell,
comprised of oligo(ethyleneoxy) chains, was weakened, re-
sulting in the deformation of the structure of the micelles.
Thus, the drug release was faster above the LCST than below
the LCST. Unfortunately, there was little change in the release
rate of POD from G3 micelles as a function of temperature,
probably because the drug would be retained in the hydro-
phobic aggregates above the LCST. Therefore, this may not
be an ideal system for thermally triggered drug release. How-
ever, it is expected that a less hydrophobic drug may be more
readily released from G3 micelles above the LCST.
Experimental
Instruments and materials
1H NMR and 13C NMR spectra were determined on a
Bruker DRX-500 with TMS as internal standard. ESI-MS
spectra were obtained on a Finnigan LCQ-Advantage mass
spectrometer. Matrix-assisted laser desorption ionization
(time of flight) mass spectrometry (MALDI-TOF MS) was
carried out on a Bruker Biflex III MALDI-TOF spectrometer
with a-cyano-4-hydroxylcinnamic acid (CCA) as the matrix.
The GPC system consisted of a Waters 2690D separations
module and a Waters 2414 refractive index detector (RI).
Styragel HR1 THF and HR2 THF columns (Waters) were
used at 40 °C with polystyrene as the standard and tetrahy-
drofuran (THF) as the mobile phase at a flow rate of
0.3 mL/min. FT-IR spectra were taken on a Nicolet AVATAR
360 FT-IR spectrometer.
Methyl
3,4,5-tris(3,4,5-tris(2-(2-methoxyethoxy)ethoxy)
benzyloxy) benzoate (G2a) was synthesized according to the
method previously reported.31 Methyl gallate was purified by
recrystallization from methanol. THF was refluxed on so-
dium and distilled from sodium benzophenone. DMF and di-
chloromethane (DCM) were distilled from anhydrous calcium
chloride for drying. Other reagents were used as received.
Conclusions
Synthesis of 3,4,5-tris(3,4,5-tris(2-(2-methoxyethoxy)
ethoxy)benzyloxy) benzyl alcohol (G2b)
Dendron G2b was prepared by the reduction of G2a with
We synthesized a novel third-generation thermoresponsive
amphiphilic dendron with a hydrophobic poly(benzyl ether)
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Xu et al.
605
Fig. 5. (a) Transmittance measurements as a function of temperature for different concentrations of G3. (b) Dependence of lower critical
solution temperatures (LSCTs) on concentrations of G3 and G2a.
Fig. 6. Plots of hydrodynamic diameters (Dh) of the micelles from
G3 and G2a in aqueous solution as a function of temperature from
dynamic light-scattering (DLS) measurements.
Fig. 8. Temperature-responsive drug release from G3 micelles in
phosphate-buffered saline solutions (PBS, pH 7.4) at 20, 37, and
55 °C, respectively.
Fig. 7. In vitro release profile of podophyllotoxin from G3 and G2a
micelles at 37 °C.
After 20 h, the reaction was quenched by dropwise addition
of 10% NaOH. The resulting precipitate was filtered and the
THF was evaporated. The residue was dissolved in DCM and
washed with saturated NaCl solution. The organic phase was
dried over MgSO4. After filtration, the DCM was rotoevapo-
rated, affording G2b as a pale yellow oil, yield 80.5%. IR
(KBr) n: 3445 (m, n(OH)), 2929 (s), 2879 (s), 1591 (s,
n(Ar)), 1501 (m, n(Ar)), 1112 (s, nas(COC)). 1H NMR
(500 MHz, CDCl3) d: 6.64 (m, 8H, Ar H), 4.96–5.00 (m,
6H, CH2), 4.53 (s, 2H, CH2), 3.95–4.18 (m, 18H, CH2),
3.77–3.85 (m, 18H, CH2), 3.68–3.72 (m, 18H, CH2), 3.53–
3.55 (m, 18H, CH2), 3.36–3.38 (m, 27H, CH3). 13C NMR
(125 MHz, CDCl3) d: 152.95, 152.68, 138.19, 137.88,
133.74, 133.02, 108.23, 107.51, 107.02, 75.09, 72.65, 72.28,
72.22, 71.53, 70.84, 70.60, 69.95, 69.11, 64.73, 59.15. ESI-
MS m/z: 1513 [M + Na]+.
Synthesis of 3,4,5-tris(3,4,5-tris(2-(2-methoxyethoxy)
ethoxy)benzyloxy) benzyl chloride (G2c)
G2c was obtained by chlorination of G2b with SOCl2.
G2b (3.88 g, 2.6 mmol) was dissolved in 50 mL of dry
DCM. DMF (4.5 mL) and SOCl2 (0.93 g, 7.8 mmol) was
then added dropwise and the reaction mixture was stirred for
LAH. To a solution of G2a (2.43 g, 1.6 mmol) in dry THF
was added LAH (0.20 g, 5.2 mmol) at –5 °C. This mixture
was stirred for 0.5 h, and then warmed to room temperature.
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Can. J. Chem. Vol. 90, 2012
another 3 h. After being washed with aqueous NaHCO3 solu-
tion and saturated NaCl, the organic phase was dried over
MgSO4 and filtered, and the solvent was rotoevaporated, af-
fording G2c as a pale yellow oil, yield 92.4%. IR (KBr) n:
2929 (s), 2879 (s), 1589 (s, n(Ar)), 1499 (m, n(Ar)), 1112
concentrations. The temperature of the solutions was raised
at a constant rate (0.4 °C/min) and the optical transmittance
of the polymer solution was measured at 500 nm with a
UV–vis spectrophotometer (Cary 50, Varian). The LCST
was defined as the temperature corresponding to the initial
break points in the resulting transmittance vs temperature
curve.39
1
(s, nas(COC)). H NMR (500 MHz, CDCl3) d: 6.63–6.65 (m,
8H, Ar H), 5.00 (s, 6H, CH2), 4.46–4.49 (m, 2H, CH2),
3.98–4.19 (m, 18H, CH2), 3.77–3.87 (m, 18H, CH2), 3.65–
3.72 (m, 18H, CH2), 3.51–3.64 (m, 18H, CH2), 3.34–3.38
(m, 27H, CH3). ESI-MS m/z: 1531 [M + Na]+.
Dynamic light-scattering (DLS) experiments
The Dh of the micelles was determined by DLS as a func-
tion of temperature. The measurements were performed with
a Zetasizer ZEN 3600 instrument (Malvern, UK) using a
light-scattering apparatus equipped with a He–Ne laser. The
scattering angle was kept at 173° (backscattering), and the
wavelength in the vacuum was set as 633 nm during the
whole experiment. Solutions of dendrons G3 and G2a in de-
ionized water (1.0 mg/mL) were filtered with a 0.45 µm filter
prior to use. Three replicates were performed for each meas-
urement and an average value was obtained.
Synthesis of methyl 3,4,5-tris(3,4,5-tris(3,4,5-tris(2-(2-
methoxyethoxy)ethoxy)benzyloxy)benzyloxy)benzoate
(G3)
G3 was obtained by the alkylation of methyl gallate with
G2c. A mixture of methyl gallate (0.09 g, 0.5 mmol), G2c
(2.41 g, 1.6 mmol), and K2CO3 (0.69 g, 5.0 mmol) in dry
DMF was stirred and heated to 70 °C for 24 h under a nitro-
gen atmosphere. After filtration and then evaporation of
DMF in vacuo, the residue was dissolved in a small amount
of distilled water, which was dialyzed against distilled water
for 3 days. The water in the dialyzed solution was removed
under reduced pressure, affording G3 as a pale yellow oil,
yield 62.3%. IR (KBr) n: 2937 (m), 2878 (m), 1723 (m,
Drug loading and in vitro release
Dendron G2a or G3 (50 mg) and POD (15 mg) were dis-
solved in 10 mL of acetone. Deionized water (10 mL) was
added dropwise to the solution under vigorous stirring. The
solution was stirred in an open air system overnight to re-
move acetone by evaporation. The precipitate of unbound
POD was removed by centrifugation at 4000 rpm for
30 min. The amount of POD entrapped in the dendron mi-
celles was determined by measuring the absorbance at
292 nm, using a standard calibration curve experimentally
obtained with POD–ethanol solutions. The DLCand EE were
calculated from the following equations:37 DLC (wt %) =
100(mass of POD in micelles / mass of POD-loaded mi-
celles); EE (%) = 100(mass of POD in micelles / mass of
POD initially loaded).
To study in vitro drug release, a dialysis bag (molecular
weight cut off = 3500 Da) containing 15 mL of the drug-
loaded micelles in suspension was immersed in a phosphate
buffer solution (150 mL, 0.01 mol/L, pH 7.4) at 20, 37, or
55 °C. At predetermined time intervals, 2 mL aliquots of the
aqueous solution were withdrawn from the release media for
UV–vis analysis, and the same volume of a fresh buffer sol-
ution was added. The amount of drug released from micelles
at different temperatures was measured by UV absorbance at
292 nm.
1
n(C=O)), 1589 (m), 1503 (m), 1110 (s, nas(COC)) H NMR
(500 MHz, CDCl3) d: 6.61–6.66 (m, 26H, Ar H), 4.94–5.07
(m, 24H, CH2), 3.92–4.13 (m, 54H, CH2), 3.78–3.79 (m,
57H, CH2 + CH3), 3.68–3.71 (m, 54H, CH2), 3.52–3.55 (m,
54H, CH2), 3.35–3.38 (m, 81H, CH3). 13C NMR (125 MHz,
CDCl3) d: 166.72 (C=O), 154.66, 153.64, 153.02, 152.79,
151.85, 149.46, 142.49, 141.52, 138.31, 136.42, 133.74,
133.02, 132.71, 131.85, 131.65, 110.19, 109.60, 107.46,
107.05, 75.86, 75.33, 73.27, 72.89, 72.70, 72.34, 72.26,
70.87, 70.66, 69.99, 69.15, 60.46, 59.20, 58.29, 52.50.
MALDI-TOF MS m/z calcd for C227H350O95: 4599; found:
4624 [M + Na]+. Anal. calcd for C227H350O95: C 59.28, H
7.67; found: C 59.01, H 7.81.
Fluorescence measurements
Fluorescence spectra were obtained on a Hitachi F-4500
luminescence spectrophotometer using pyrene as a fluores-
cent probe. Aliquots of pyrene solution (5.9 × 10–6 mg/mL
in acetone, 50 µL) were added to volumetric flasks and the
acetone was evaporated overnight. Then, aqueous solutions
of G3 at different concentrations were added to the flasks.
The final pyrene concentration in each sample was 5.9 ×
10–7 mg/mL. The solutions were kept at room temperature
for 24 h to equilibrate the pyrene and the micelles. The
emission spectra were recorded from 250 to 500 nm with
an excitation wavelength of 390 nm.
Supplementary data
Supplementary data are available with the article through
the journal Web site http://nrcresearchpress.com/doi/suppl/
10.1139/v2012-038.
TEM measurements
A drop of aqueous dendron solution (1 mg/mL) was
placed on a Formwar-coated copper grid and dried in air.
TEM observation of the micelles was conducted on a Hitachi
H-600 instrument operating at an acceleration voltage of
80 kV.
Acknowledgements
The authors gratefully acknowledge support for this work
of the National Natural Science Foundation of China (Grant
No. 20864003), the Program for Changjiang Scholars and In-
novative Research Team in University (PCSIRT), and the
Natural Science Foundation of Yunnan Province (Grant No.
2009ZC064M). The authors also thank Professor Wenfu Fu
for assistance with MALDI-TOF MS measurements.
LCST measurement
The LCST was determined by cloud point measurement.
Dendron G3 was dissolved in deionized water at different
Published by NRC Research Press

Xu et al.
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