Facile synthesis of polyester dendrimers as drug delivery carriers

Article abstract of DOI:10.1021/ma301849a

Aliphatic polyester dendrimers are attractive carriers for in vivo delivery of bioactive molecules due to their biocompatibility and biodegradability, but efficient precision synthesis of these dendrimers without tedious purifications remains challenging. Herein, we report an efficient synthesis approach to polyester dendrimers from two AB₂-type monomers via combining a click reaction of thiol/acrylate Michael addition with esterification. The reaction solution of each generation contains only the targeted dendrimer macromolecules; thus, the only required separation is simple precipitation. The resulting hydroxyl-terminated fifth-generation dendrimer is thermoresponsive with a LCST of 41 C. The dendrimer could be further pegylated to obtain a water-soluble biocompatible dendrimer capable of encapsulation and controlled release of a hydrophobic anticancer drug, doxorubicin.

Full text of DOI:10.1021/ma301849a

Article
pubs.acs.org/Macromolecules
Facile Synthesis of Polyester Dendrimers as Drug Delivery Carriers
Xinpeng Ma, Zhuxian Zhou, Erlei Jin, Qihang Sun, Bo Zhang, Jianbin Tang, and Youqing Shen*
‡
‡
‡
‡
‡
†
,†
†Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Center for Bionanoengineering, and Department of
Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, China
‡
Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, Wyoming 82071, United States
ABSTRACT: Aliphatic polyester dendrimers are attractive
carriers for in vivo delivery of bioactive molecules due to their
biocompatibility and biodegradability, but efficient precision
synthesis of these dendrimers without tedious purifications
remains challenging. Herein, we report an efficient synthesis
approach to polyester dendrimers from two AB -type
2
monomers via combining a click reaction of thiol/acrylate
Michael addition with esterification. The reaction solution of
each generation contains only the targeted dendrimer
macromolecules; thus, the only required separation is simple
precipitation. The resulting hydroxyl-terminated fifth-gener-
ation dendrimer is thermoresponsive with a LCST of 41 °C.
The dendrimer could be further pegylated to obtain a water-soluble biocompatible dendrimer capable of encapsulation and
controlled release of a hydrophobic anticancer drug, doxorubicin.
INTRODUCTION
some characteristics of click reaction and have also been used to
14,15
■
synthesize dendrimers.
For instance, bis-MPA was
Dendrimers are highly branched macromolecules characterized
by monodispersity, uniform and controlled sizes, copious
functionalized with allyl or acetylene groups and was used for
16
1
2
dendrimer synthesis via thiol−ene reaction. Very recently,
surface functionalities, and low intrinsic viscosity in solution.
Hawker and Malkoch introduced thiol and azides or acetylene
These characteristics make them ideal nanocarriers for
1
a,3
and allyl groups to bis-MPA separately and obtained AB
CD monomers. Alternative azide−acetylene and thiol−ene
click reactions produced dendrimers quickly and efficiently.
2
and
biomedical applications,
PAMAM) dendrimers are the most studied. However,
PAMAM dendrimers are not biodegradable in vivo and carry
of which polyamidoamine
4
2
(
17
4
a,5
However, the radical nature of thiol−ene reactions caused
cross-linking due to radical coupling, particularly in the
synthesis of dendrimers higher than fifth generations, causing
broader polydispersity (PDI > 1.2).
positive charges on their surface, thus inducing cytotoxicity,
6
6
hemolytic toxicity,7 rapid blood clearance, and quick
opsonization (RES). These drawbacks hinder their translation
to clinical applications. Aliphatic polyester dendrimers, for
Aliphatic polyester dendrimers without heterocyclics have
better biocompatibility and biodegradability for translational
nanocarriers. Taking advantage of highly efficient thiol/acrylate
Michael addition reactions, we developed a simple but efficient
strategy to synthesize bis-MPA-based dendrimers without any
protection/deprotection steps. The monomers were easily
obtained and the reactions were fast under mild conditions. A
dendrimer with 128 terminal hydroxyl groups was constructed
in five steps (Scheme 1) with a high overall yield. We also
demonstrated an application of the dendrimer as a drug carrier.
example the dendrimers from an AB -type monomer 2,2-
2
bis(hydroxymethyl)propionic acid (bis-MPA), are biodegrad-
able and biocompatible with very low toxicity and low
8
immunogenicity and thus have been proposed as carriers for
9
in vivo biodelivery or imaging.
The traditional polyester dendrimers were synthesized by
repeated esterification of bis-MPA with protected either
10
11
carboxylic acid group or hydroxyl groups followed by
deprotection. These synthesis techniques are straightforward,
but the protection/deprotection reactions may be incomplete
and thus introduce defects amplified in the subsequent
generations and also make the synthesis tedious.
EXPERIMENTAL SECTION
■
The azide/acetylene-based click reaction characteristic of
Materials. 2,2-Bis(hydroxymethyl)propionic acid (bis-MPA, 98%),
pentaerythritol (≥99%), N,N′-diisopropylcarbodiimide (DIC, 99%),
acryloyl chloride (98%), triethylamine (Et N, 99.5%), sodium
12
specificity, quantitative yields, and almost perfect fidelity has
been used extensively in synthesis of dendrimers with fewer
3
13
carbonate (≥99%), 1-thioglycerol (99%), succinic anhydride
steps, less purification procedures, and higher overall yields.
For instance, acetylene or azide groups were separately
introduced to bis-MPA to produce asymmetric clickable
monomers used for speed synthesis of dendrimers. The
Received: September 3, 2012
Revised: November 28, 2012
Published: December 18, 2012
14
thiol/allyl- or thiol/acetylene-based thiol−ene reactions have
©
2012 American Chemical Society
37
dx.doi.org/10.1021/ma301849a | Macromolecules 2013, 46, 37−42

Macromolecules
Article
Synthesis of 2,2-Bis(acryloyloxymethyl)propionic Acid
Scheme 1. Dendrimer Synthesis from a AB Monomer Pair
2
(
ACPA). Bis-MPA (30 g, 0.22 mol) in 300 mL of dichloromethane,
2,2-Bis(acryloyloxymethyl)propionic Acid (ACPA) and 1-
DMAP (1.40 g), and TEA (77 mL) were charged to a 1 L flask and
cooled to 0 °C. Acryloyl chloride (38.5 mL, 0.47 mol) was added
dropwise to the solution for 3 h with stirring. The mixture was
extracted with a Na CO (10%) aqueous solution. The aqueous phase
Thioglycerol
2
3
was acidified with concentrated hydrochloride acid and then extracted
with dichloromethane. The organic phase was dried with sodium
sulfate, and then the solvent was evaporated. The crude product was
further purified with column chromatography (hexane/ethyl acetate
1
5
:1) to obtain pure ACPA acid as a white powder (21.7 g). H NMR
(
5
CDCl , 400 MHz): δ : 6.41 (d, J = 17.6 Hz, 2H), 6.09 (m, 2H),
.86 (d, J = 11.2 Hz, 2H), 4.37 (s, 4H), 1.33 (s, 3H). C NMR
3 ppm
1
3
(
CDCl , 400 MHz): δppm: 178.34, 165.65, 131.63, 127.67, 65.26,
3
4
6.14, 17.69.
Synthesis of Pentaerythritol Tetraacrylate (PTA). Pentaery-
thritol (0.5 g, 3.67 mmol) was suspended in a solution of triethylamine
2.5 mL, 18 mmol) and dichloromethane (10 mL) and was cooled to
°C. Acryloyl chloride (1.30 mL, 16.1 mmol) was added dropwise to
(
0
the solution in 0.5 h with stirring, and the reaction mixture was stirred
for 2 h at room temperature. The mixture was then washed with a
Na CO (10%) aqueous solution. The organic phase was dried with
2
3
Na SO , and then the solvent was evaporated to give the crude
2
4
product. It was further purified with column chromatography (hexane/
ethyl acetate 10:1) to give the PTA as a colorless oil with a 96.6% yield
1
(
4
1.25 g). H NMR (CDCl , 400 MHz): δ : 6.44 (d, J = 17.6 Hz,
3
ppm
1
3
H), 6.11 (m, 4H), 5.89 (d, J = 10.4 Hz), 4.29 (s, 8H). C NMR
(
CDCl , 400 MHz): δppm: 165.06, 131.32, 127.76, 62.45, 41.96.
Synthesis of G1-8OH. 1-Thioglycerol (1.56 g, 14.42 mmol) was
3
added to the solution of PTA (0.85 g, 2.41 mmol) and Et N (0.24 g,
3
2
.39 mmol) in DMSO (4 mL). The solution was stirred at room
temperature for 0.5 h and then diluted with methanol (4 mL). The
mixture was poured into ether (30 mL). The product was isolated and
purified by reprecipitation in ether. The G1-8 was obtained as a
OH
1
colorless oil (1.81 g) at a yield of 95.9%. H NMR (DMSO-d , 400
6
MHz): δppm: 4.76 (d, J = 4.8 Hz, 4H), 4.57 (t, J = 4.8 Hz, 4H), 4.09 (s,
8
7
H), 3.54 (m, 4H), 3.32 (m, 8 H), 2.71 (d, J = 6.8 Hz, 8H), 2.62(t, J =
.2 Hz, 8H), 2.45 (t, J = 7.2 Hz, 8H). 13C NMR (DMSO-d , 400
6
MHz): δppm: 171.59, 71.92, 64.91, 62.56, 35.47, 34.80, 22.51. MS
(
8
MALDI-TOF, m/z) Calcd for C H O S : 784.2138; found:
08.4278 (M + Na ); found: 824.4160 (M + K ). GPC: M , 830
n
29 52 16 4
+
+
Da; PDI: 1.003.
Synthesis of G2-16acrylate. G1-8OH (0.50 g, 0.64 mmol), ACPA
(3.67 g, 15.29 mmol), DIC (2.83 mL, 18.35 mmol), and DMAP (0.22
g, 1.81 mmol) were dissolved in 10 mL of dichloromethane. The
solution was stirred overnight at room temperature. After evaporation
of the solvent, ether was added to the residue and filtered. The filtrate
was precipitated in hexane (50 mL × 3), and the precipitation was
(
(
≥99%), 4-(N,N-dimethylamino)pyridine (DMAP, ≥99%), poly-
ethylene glycol) monomethyl ether (PEG2k, MW 2000 Da), and
doxorubicin hydrochloride (DOX·HCl, ≥98%) were purchased from
Sigma-Aldrich (Milwaukee, WI). Hydrochloric acid (37%), anhydrous
dichloromethane (99.96%), ethyl ether (99.0%), methanol (99.0%),
tetrahydrofuran (THF, ≥99.0%), and hexane (98.5%) were from
Fisher Scientific (Pittsburgh, PA). Anhydrous dimethyl sulfoxide
dried in the vacuum to obtain the product G2-16acrylate as colorless oil
1
(
DMSO, 99.9%) was from EMD Chemicals (Gibbstown, NJ). All
(1.54 g) at a yield of 90.1%. H NMR (CDCl
3
, 400 MHz): δppm: 6.37
chemicals were used as received.
(d, J = 17.2 Hz, 16H), 6.09 (m, 16H), 5.83 (d, J = 8.4 Hz, 16H), 5.13
(m, 4H), 4.43−4.12 (m, 48H), 2.76 (t, J = 6.8 Hz, 8H), 2.67 (t, J = 6.8
Instrumentation. Gel permeation chromatography (GPC) was
performed on a Waters SEC equipped with a Waters 2414 refractive
index detector, a PD2000 dynamic laser light scattering detector with
Hz, 8H), 2.59 (t, J = 6.8 Hz, 8H), 1.25 (s, 24H). 13C NMR (CDCl
,
3
400 MHz): δppm: 172.13, 171.90, 170.90, 165.72, 165.40, 165.37,
131.65, 131.62, 131.60, 131.57, 130.98, 128.15, 127.77, 127.71, 70.93,
65.24, 64.18, 62.13, 46.51, 46.48, 42.11, 34.24, 31.89, 27.05, 17.76. MS
1
5° and 90° scattered light collecting angles, and two 300 mm Solvent-
2 4
Saving GPC Columns (molecular weight ranges: 5 × 10 −3 × 10 , 5 ×
1
3
5
0 −6 × 10 ) set at 30 °C. THF with 3% v/v Et N was used as eluent
(MALDI-TOF, m/z) Calcd for C117
H
148
O
56
S
4
: 2576.7616; found:
, 2600 Da; PDI: 1.031.
Synthesis of G3-32OH. Reaction of G2-16acrylate (0.40 g, 0.16 mmol)
with 1-thioglycerol (0.66 g, 6.11 mmol) in the presence of Et N (0.15
3
+
at a flow rate of 0.30 mL/min. Data were recorded and processed
using the Waters software package. Nuclear magnetic resonance
2599.6706 (M + Na ). GPC: M
n
(
NMR) spectra were recorded on a Bruker Avance DRX-400
3
spectrometer using CDCl or DMSO-d as solvent. Chemical shifts
mL, 1.05 mmol) in DMSO (5 mL) following the procedure used in
3
6
were reported downfield from 0.00 ppm using TMS as an internal
reference. Matrix-assisted laser-desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS; Applied Biosystems, Voyager
DE Pro) was performed in a positive-ion mode with a source
temperature of 200 °C at a sample concentration of about 100 μM
the G1-8OH synthesis produced G3-32OH as a colorless oil (0.61 g) with
1
a 94.9% yield. H NMR (DMSO-d
, 400 MHz): δppm: 5.11 (b, 4H),
6
4.75 (b, 32H), 4.60 (b, 32H), 4.16 (b, 48H), 3.66 (m, 16H), 3.54 (m,
32H), 2.71 (t, J = 6.8 Hz, 48H), 2.59 (t, J = 7.2 Hz, 48H), 2.46 (t, J =
6.8 Hz, 20H), 1.18 (s, 24H). 13C NMR (DMSO-d , 400 MHz): δppm:
6
using 2,5-dihydroxybenzoic acid as the matrix. The size of the G5-PEG
/
172.37, 172.09, 171.53, 171.41, 71.84, 70.66, 70.64, 65.32, 64.96,
64.89, 55.27, 46.43, 46.40, 43.53, 43.49, 35.46, 34.75, 27.48, 17.77,
17.70. MS (MALDI-TOF, m/z) Calcd for C165H276O S : 4305.1536;
DOX was determined using a Nano-ZS Nanosizer (Malvern
Instruments, Worcestershire, UK) with a laser wavelength of 632.8
nm and a scattering angle of 173°.
88
20
+
found: 4325.9125 (M + Na ). GPC: M , 4400 Da; PDI: 1.036.
n
3
8
dx.doi.org/10.1021/ma301849a | Macromolecules 2013, 46, 37−42

Macromolecules
Article
Synthesis of G4-64acrylate. Reaction of G3-32OH (0.09 g, 0.021
mmol) with ACPA (0.80 g, 3.33 mmol) in the presence of DIC (0.62
mL, 3.99 mmol) and DMAP (0.05 g, 0.41 mmol) in CH Cl (5 mL)
dialysis bag (Spectra Por-7, molecular weight cutoff, MWCO, 3500)
and dialyzed against pH 8.5 buffer for 24 h. The DOX content was
analyzed by measuring its UV−vis absorbance at 486 nm in DMSO
against a standard curve constructed with DOX solutions of known
concentrations. The average diameter of G5-PEG/DOX measured by
DLS was about 19.5 nm.
2
2
following the procedure used in the G2-16acrylate synthesis produced
1
G4-64Acrylate as a colorless oil (0.22 g) at a 91.6% yield. H NMR
(
CDCl , 400 MHz): δppm: 6.40 (d, J = 17.2 Hz, 64H), 6.09 (m, 64H),
3
5
.83 (d, J = 10.4 Hz, 64H), 5.13 (b, 20H), 4.46−4.17 (m, 208H), 2.78
Measurement of the Drug Release of G5-PEG/DOX. G5-PEG
loaded with 15.2 wt % DOX (3.8 mg in 10 mL of buffer solution) was
loaded in a dialysis bag (MWCO 3500, Spectrum) and incubated in
100 mL of phosphate buffer saline (PBS, pH 5.0 or 7.4) at 37 °C in a
water bath with shaking. At timed intervals, 10 mL of the solution was
taken from outside the dialysis bag and 10 mL fresh PBS was added.
The DOX concentration was determined by UV−vis absorbance at
486 nm, and the percentage of DOX released was calculated.
In Vitro Cytotoxicity Assay. The cytotoxicity assay was carried
out using the MTT cell-proliferation assay kit (ATCC, Manassas, VA)
according to the modified manufacturer’s protocol. SKOV-3 ovarian
cancer cells were cultured in a medium (Invitrogen Corp., Carlsbad,
CA) for at least 2 weeks before use. They were then seeded onto 96-
well plates at a density of 10 000 cells per well and incubated for 72 h.
(
t, J = 6.4 Hz, 40H), 2.70 (t, J = 6.4 Hz, 40H), 2.59 (t, J = 6.8 Hz,
4
1
6
0H), 1.25 (s, 120H). 13C NMR (CDCl , 400 MHz): δppm: 172.17,
3
71.91, 170.94, 165.42, 131.67, 131.64, 128.10, 127.81, 127.74, 70.98,
5.26, 64.26, 46.53, 46.51, 34.26, 31.96, 27.09, 17.79. MS (MALDI-
TOF, m/z) Calcd for C517H660O248S20: 11475.3448; found: 11
+
500.5951 (M + Na ). GPC: M , 12 000 Da; PDI: 1.044.
n
Synthesis of G5-128OH. Reaction of G4-64acrylate (0.40 g, 0.035
mmol) with 1-thioglycerol (1.92 g, 17.8 mmol) in the presence of
Et N (0.32 mL, 2.30 mmol) in DMSO (5 mL) following the
procedure used in the G1-8OH synthesis produced G5-128OH as a
colorless oil (0.58 g) at a 90.6% yield. H NMR (DMSO-d , 400
MHz): δppm: 5.14 (b, 20H), 4.76 (b, 128H), 4.57 (b, 128H), 4.17 (b,
2
2
3
1
6
08H), 3.55 (m, 128H), 3.34 (m, 256 H), 2.72 (d, J = 6.8 Hz, 168H),
13
.62 (m, 168H), 2.45 (t, J = 6.8 Hz, 168H), 1.24 (s, 120H). C NMR
The original medium (200 mL) was replaced with the G5−PEG,
(
7
3
DMSO-d , 400 MHz): δppm: 173.56, 172.36, 172.09, 171.52, 171.39,
1.89, 71.21, 65.28, 64.92, 49.07, 48.55, 46.40, 36.27, 35.49, 34.76,
G5−PEG/DOX, or free DOX·HCl solutions at different concentrations.
The cells were incubated for 72 h, and then the medium in each well
was replaced with fresh cell culture medium and further incubated for
48 h. MTT reagent (10 mL) was then added to each well and
incubated for 6 h. Finally, the detergent reagent (100 mL) was added
to each well, and the plates were incubated at 37 °C for 18 h to
dissolve the crystals. The absorbance intensity at 570 nm was
recorded, and the cytotoxicity was expressed as a percentage of the
control.
6
4.42, 31.51, 27.50, 26.87, 17.82, 17.73, 17.38. GPC: M , 19 400 Da;
n
PDI: 1.052. The diameter of G5-128OH measured by DLS in water (1
mg/mL) was 5.20 ± 0.10 nm.
Synthesis of PEG2k-COOH. PEG2k (10 g), succinic anhydride (2
g, 20 mmol), and DMAP (1.22 g, 10 mmol) were dissolved in 40 mL
of CH Cl and stirred at room temperature for 24 h. The solvent was
removed under reduced pressure, and then the residue was dissolved
in 50 mL of deionized (DI) water. The product was extracted with
CH Cl (30 mL × 3), and the organic layer was dried with sodium
sulfate. After removing the solvent under vacuum, the product was
obtained as a white solid (9.3 g). H NMR (CDCl , 400 MHz): δ
4
NMR (CDCl , 400 MHz): δ : 173.18, 171.85, 71.58, 70.22, 68.66,
6
2
2
RESULTS AND DISCUSSION
2
2
■
The hydroxyl groups in bis-MPA must be protected first to
avoid self-esterification or convert to other functional groups
1
:
C
3
ppm
1
3
.26 (m, 2H), 3.74−3.47 (m, 180H), 3.46 (s, 3H), 2.66 (m, 4H).
11b,18
that cannot cause cross-linking.
A monomer pair of
3
ppm
thioglycerol (AB ) and ACPA (CD ) simplified the reaction
3.40, 58.59, 58.56, 53.89, 53.85, 28.73, 28.35. GPC: M , 2200 Da;
2
2
n
PDI: 1.052.
requiring no protection/deprotection steps. The Michael
addition reaction of thiol−acrylate is almost quantitative
without side reactions and considered to be a click reaction
Synthesis of Pegylated G5 (G5-PEG). G5-128OH (44 mg, 2.38
μmol), PEG2k-COOH (0.96 g, 0.46 mmol), DIC (0.08 mL, 0.52
mmol), and DMAP (0.02 g) were dissolved in THF (5 mL) and
stirred overnight at room temperature. The filtrate was precipitated in
a solvent of THF and ether (v/v 1:1). Precipitate was dried under
vacuum for 6 h, and the product was obtained as a white powder (130
19
20
in polymer synthesis and functionalization. Different from
the radical mechanism of the thiol−ene/yne reactions, the
thiol−(meth)acrylate reaction does not involve radicals,
15a,21
avoiding side reactions via radical coupling.
1
mg). H NMR (CDCl , 400 MHz): δ : 5.18 (b), 4.22 (m), 3.63
3
ppm
13
The PTA was first reacted with the thiol group in
thioglycerol to produce the first-generation dendrimer with
eight hydroxyl groups (Scheme 1, step i). Pendant hydroxyl
groups were esterified with ACPA with catalysis of DIC/DMAP
(Scheme 1, step ii). Alternating the two steps easily produced
the fifth generation of the dendrimers at high overall yields
(
m), 3.47 (s), 2.81 (m), 2.61 (m), 1.27 (b). C NMR (CDCl , 400
3
MHz): δppm: 172.27, 172.10, 171.79, 171.53, 171.02, 170.55, 170.31,
7
5
2
1
1.83, 71.76, 71.71, 70.47, 69.15, 68.93, 68.52, 67.10, 66.49, 63.78,
8.95, 49.83, 46.05, 35.71, 34.46, 34.30, 32.40, 31.96, 30.64, 28.94,
8.88, 28.79, 28.73, 27.36, 27.20, 26.27, 25.39, 25.27, 24.67, 17.86,
1
7.82, 17.74. GPC: M , 145 500 Da; PDI: 1.061. The H NMR
n
spectrum showed that on average each G5 molecule was conjugated
with about 56 PEG2k chains (G5-PEG). The average diameter of
G5−PEG in water (1 mg/mL) measured by DLS was about 12.2 nm.
Determination of the Lower Critical Solution Temperature
(
68%). The synthesis strategy is shown in Scheme 1.
The reaction between PTA and a slight excess of 1-
thioglycerol in the presence of a catalytic amount of the
triethylamine was carried out at room temperature. Completion
of G5-128OH and G3-
. The lower critical solution temperatures
32OH
1
of the acrylate groups’ reaction was confirmed by H NMR and
(
LCSTs) of the G1-8OH, G3-32OH, and G5-128OH were detected using a
1
MALDI-TOF mass spectra. The H NMR spectrum of the
cloud point method. In brief, the dendrimers were dissolved in DI
water at different concentrations. The dendrimer solution was
equilibrated for 5 min at a set temperature controlled by a RC20
thermostat (Brinkmann, Dallas, TX). Transmittance through the
aqueous dendrimer solution at the wavelength of 500 nm was recorded
using a UV−vis spectrometer (UV-1201, Shimadzu, Japan). The
transmittance was plotted versus temperature, and the LCST was
defined as the midpoint of the transition.
Loading DOX to the G5-PEG Dendrimer. Doxorubicin hydro-
chloride salt (4 mg) was dissolved in 5 mL of DMSO, and two drops
of triethylamine were added to the solution. G5-PEG (20 mg) was
dissolved in 10 mL of DI water and dropped in the DOX solution. The
mixture was stirred at room temperature for 3 h and then loaded into a
reaction mixture showed that the signals at 5.8−6.4 ppm
(Figure 1a) assigned to the acrylate protons disappeared,
indicating a quantitative reaction (Figure 1b). The MALDI-
TOF MS spectrum of the reaction solution confirmed that the
targeted moleculestheoretical MW 784; found 807 (M +
+
+
Na ) and 824 (M + K )were the only products in the
solution (Figure 2).
Simple precipitation of the solution in ethyl ether removed
the unreacted 1-thioglycerol, yielding the pure first generation
(G1-8OH). G1-8OH was then reacted with ACPA catalyzed by the
DIC/DMAP coupling agents. The esterification of G1 was
3
9
dx.doi.org/10.1021/ma301849a | Macromolecules 2013, 46, 37−42

Macromolecules
Article
evaporation as a result of its high molecular weight. However,
its GPC trace was as narrow as that of the prior generation
(
Figure 2b), and DLS showed that it had a diameter of 5.2 nm
in water with a low PDI (data not shown), indicating the fifth
generation also had similar perfect structure. Thus, the reaction
solution contained only the targeted dendrimer macro-
molecules and a small amount of the unreacted monomers.
As a result, the purification required only simple precipitation.
1
The typical H NMR spectra of the acrylate- and hydroxyl-
terminated dendrimers (G4-64acrylate in CDCl and G5-
in
3
128OH
DMSO-d ) are shown in Figure 3.
6
1
Figure 1. H NMR spectra of PTA (a) and its reaction with 1-
thioglycerol in DMSO (thiol/acrylate molar ratio of 1.5, room
temperature, 0.5 h) (b).
1
Figure 3. H NMR spectra of G4-6
DMSO-d6).
(in CDCl ) and G5- (in
128OH
4acrylate
3
The hydroxyl-terminated dendrimers were water-soluble at
room temperature. G1-8OH and G3-32OH remained water-soluble
at a high concentration (up to 5 wt %) at high temperatures
(
up to 80 °C) (Figure 4). Upon increasing the temperature
Figure 2. Molecular-weight progress of the dendrimers from the
reaction of ACPA and thioglycerol measured by (a) MALDI-TOF MS
and (b) GPC. The MALDI-TOF MS spectra were obtained from the
reaction solutions without any purification.
monitored using MALDI-TOF analysis to ensure completion.
We found that a three-to-one ratio of ACPA relative to each
hydroxyl group (COOH/OH = 3) was needed to complete the
esterification and produce the target G2-16acrylate (Figure 2a).
DCC and DMAP also catalyzed this reaction to completion
under the same conditions, but DCC and its byproducts were
difficult to remove from the product. DIC and G2-16acrylate are
soluble in ether, but the product of N,N′-diisopropylurea is not.
Figure 4. Transmittance of the solutions of G1-8OH, G3-32OH, and
G5-128OH in DI water as a function of temperature at different
concentrations.
higher than 41 °C, the clear solution of G5-128OH dendrimer
suddenly became cloudy and the dendrimer precipitated
(Figure 4). This soluble/insoluble transition at the lower
critical solution temperature (LCST) was generally due to the
disturbance of hydrophilicity/hydrophobicity balance of the
Therefore, G2-1
was easily isolated by ether extraction
6acrylate
and precipitation.
Figure 2a shows the MALDI-TOF MS spectra of the reaction
solutions. Clearly, the reaction solution in each generation only
contained the targeted dendrimer molecules in agreement with
the calculated molecular weight. There were almost no signals
of incomplete molecules. For example, the reaction solution of
the fourth-generation dendrimer G4-64acrylate had a molecular
ion at 11 500.59, which was the sodium ion adduct with the
molecule (11 475.34; Figure 2a). The MALDI-TOF spectrum
of the fifth generation had a poor resolution due to difficult
22
polymer chains. The average hydrophobic segment per
hydroxyl group was 126.7 Da for G5-128OH but 98.0 Da for
G1-8OH and 117.5 Da for G3-32OH. Therefore, G5-128OH was
more hydrophobic than G1-8OH and G3-32OH; upon heating
some hydroxyl groups were dehydrated, further increasing the
overall hydrophobicity of the dendrimer and leading to
precipitation. This phenomenon was consistent with our recent
22b
results of thermally responsive polyester dendrimers
and
4
0
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Macromolecules
Article
2
3
other reports. Interestingly, this phenomenon was opposite to
the conclusion reported by Adronov et al. that the bis-MPA
polyester dendrimers functionalized with carborane had a
LCST, but the parent dendrimers exhibited no phase
2
4
transition.
An important application of this type of dendrimer is as a
drug carrier owing to its hydrophobic interior. To demonstrate
this concept, PEG2 kDa chains were first introduced onto the
dendrimer surface of G5-128OH to further grant it stealth
properties. The esterification of G5-128OH using PEG2k-COOH
in the presence of DIC and DMAP (Scheme 2) introduced a
Figure 6. Cytotoxicity of free DOX, G5-PEG/DOX (15.2 wt % DOX)
(a) and G5-PEG (b) to SKOV-3 ovarian cancer cells estimated by MTT
assay. Cells were exposed to the indicated drug or polymer for 72 h in
medium. Data represent mean ± sd, n = 5.
Scheme 2. Synthesis of G5-PEG and Its Loading with DOX
−1
to SKOV-3 ovarian cancer cells was 0.085 μg mL , no
−1
significant difference from that of free DOX (0.056 μg mL ).
CONCLUSION
■
In summary, we successfully developed an efficient synthesis of
monodispersed bis-MPA polyester dendrimers using thiol−
acrylate reaction and the traditional esterification reaction
under mild conditions. The 64-acrylate-terminated dendrimer
was obtained in four steps, and the 128-hydroxyl-terminated
dendrimer was produced in five steps. The simple synthesis and
purification make the dendrimer synthesis straightforward for
large-scale production. The hydroxyl-terminated dendrimers
were thermoresponsive, and the LCST was 41 °C, which is near
the physiological temperature. The biocompatible dendrimer
G5-PEG showed an excellent capacity for the encapsulation and
controlled release of a hydrophobic anticancer drug such as
DOX. Further applications of the dendrimers as drug carriers
are under exploration.
controlled number of PEG chains. One such sample contained
about 56 PEG chains (G5-PEG) as determined by the
integration of the methyl group (1.27 ppm) of dendrimer
1
and PEG (3.6 ppm) in its H NMR spectrum. DOX, a
hydrophobic anticancer drug, was easily encapsulated into
G5-PEG via dialysis. The DOX content was 15.2% with a loading
efficiency of 99%, which suggests that the G5-PEG accom-
modated DOX very well via the hydrophobic−hydrophobic
interaction.
As shown in Figure 5, G5-PEG/DOX released DOX with a
slight burst at pH 7.4 and 37 °C followed by a very slow
AUTHOR INFORMATION
Corresponding Author
E-mail shenyq@zju.edu.cn or sheny@uwyo.edu.
■
*
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the National Science Foundation of China
21090352), the National Fund for Distinguished Young
■
(
Scholars (50888001), the Program for Changjiang Scholars
and Innovative Research Team of the University of China, and
the U.S. Department of Defense (BC090502) for financial
support.
Figure 5. DOX release from G5-PEG/DOX at pH 7.4 (▲) or 5 (◆)
and 37 °C as a function of time.
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