Amphiphilic triblock copolymer bioconjugates with biotin groups at the junction points: Synthesis, self-assembly, and bioactivity

Article abstract of DOI:10.1021/ma102398c

Poly(ε-caprolactone)-block-poly(ethylene glycol)-block-poly(ε- caprolactone) (PCL-b-PEG-b-PCL) triblock copolymers with biotin groups at the junction points were synthesized based on a combination of click chemistry and ring-opening polymerization (ROP). Alkyne-functionalized PCL-b-PEG-b-PCL triblock copolymers were synthesized by using alkyne-functionalized PEG as macroinitiators in ROP of ε-caprolactone. Click chemistry was employed in the synthesis of the biotinylated triblock copolymers. Gel permeation chromatography and ¹H NMR results all indicated successful synthesis of well-defined triblock copolymers. The triblock copolymer chains can self-assemble into micelles in aqueous solution. The PCL blocks form the cores of the micelles and the hydrophilic PEG blocks form the coronae. The biotin moieties distribute at the interface of the micelles. Upon addition of avidin to the micellar solution, micelles aggregated together forming micellar aggregates due to the interaction between avidin and biotin. The avidin/HABA competitive binding assay also proved the bioavailability of the biotinylated micelles to avidin.

Full text of DOI:10.1021/ma102398c

ARTICLE
pubs.acs.org/Macromolecules
Amphiphilic Triblock Copolymer Bioconjugates with Biotin Groups at
the Junction Points: Synthesis, Self-Assembly, and Bioactivity
Jie Jin,†,§ Dongxia Wu,†,§ Pingchuan Sun,‡ Li Liu,*,‡ and Hanying Zhao*,†
†Key Laboratory of Functional Polymer Materials, Ministry of Education, Department of Chemistry and ‡Institute of Polymer
Chemistry, Nankai University, Tianjin 300071, P. R. China
S Supporting Information
b
ABSTRACT: Poly(ε-caprolactone)-block-poly(ethylene glycol)-block-
poly(ε-caprolactone) (PCL-b-PEG-b-PCL) triblock copolymers with
biotin groups at the junction points were synthesized based on a
combination of click chemistry and ring-opening polymerization
(ROP). Alkyne-functionalized PCL-b-PEG-b-PCL triblock copolymers
were synthesized by using alkyne-functionalized PEG as macroinitiators
in ROP of ε-caprolactone. Click chemistry was employed in the
synthesis of the biotinylated triblock copolymers. Gel permeation
chromatography and 1H NMR results all indicated successful synthesis
of well-defined triblock copolymers. The triblock copolymer chains can
self-assemble into micelles in aqueous solution. The PCL blocks form
the cores of the micelles and the hydrophilic PEG blocks form the
coronae. The biotin moieties distribute at the interface of the micelles.
Upon addition of avidin to the micellar solution, micelles aggregated
together forming micellar aggregates due to the interaction between
avidin and biotin. The avidin/HABA competitive binding assay also
proved the bioavailability of the biotinylated micelles to avidin.
’ INTRODUCTION
Biotin is a biomolecule that can bind to the enzyme avidin, yielding
a stable complex with an association constant of 1015 mol-1.13 Avidin
has four binding sites for biotin and can serve as a linkage between the
biotinylated materials and other proteins. Recently, the biotin-avidin
complex system was introduced to drug delivery systems for pre-
targeting purposes.14 Biotinylated polymers have been known to
undergo receptor-mediated endocutosis in both animal and plant
cells.15 Biodegradable biotinylated homopolymers16 and block copo-
lymers were synthesized by various approaches.17 The Cu(I)-
catalyzed Huisgen 1,3-dipolar cycloaddition reactions of azides and
organic alkynes, also called “click” chemistry, were introduced by
Sharpless et al.18 Click reactions have gained a great deal of attention
due to the excellent functional-group tolerance, high specificity, and
nearly quantitative yields under mild experimental conditions.19,20
Click reactions have been widely used as a versatile method in the
synthesis of polymers with different architectures,21-24 monomers,25
and protein-based biohybrids.26-28 In previous papers we reported
preparation of biotinylated polymeric micelles by postpolymerization
functionalization29 and preparation of biotinylated Janus silica parti-
cles by in-situ click chemistry.30
Covalent conjugation to synthetic polymers is an efficient way to
improve the stability, activity, and biocompatibility of many bioma-
cromolecules. Bioconjugate polymers present virtues of both syn-
thetic polymers and biomacromolecules and have found a variety of
applications, including drug delivery,1 biocatalysis,2 and biosensing.3
Biodegradable and biocompatible aliphatic polyesters have re-
ceived great attention over the past two decades. Among them,
poly(ε-caprolactone) (PCL), poly(lactic acid), and their copoly-
mers have been widely developed in medical reaserch.4-7 PCL is a
nontoxic biodegradable polymer, which can be biodegraded in the
Krebs cycle into soluble nontoxic oligomers.8 In the meanwhile,
PCL is a hydrophobic and crystalline polymer, which considerably
restrains its potential applications. Introduction of hydrophilic
polyether blocks into PCL chains is a means to enhance the
hydrophilicity as compared with the parent homopolymer. Poly-
(ethylene glycol) (PEG) is an uncharged, water-soluble, nontoxic,
nonimmunogenic polymer and therefore an ideal material for
bioapplications. In block copolymers of PEG and PCL (PEG-b-
PCL), the PCL blocks present hydrophobic properties while PEG
blocks present hydrophilic properties in the final materials. So block
copolymers of PEG and PCL have been developed for their unique
properties, such as biocompatibility, amphiphilicity, self-assembly,
permeability, biodegradability, and crystallization properties.9-12
But until now, the study on functionalization of PEG and PCL
block copolymers is limited.
In this paper, a combination of ring-opening polymerization
(ROP) and 1,3-dipolar cycloaddition reactions of azides and
Received: October 21, 2010
Revised:
February 16, 2011
Published: March 07, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Biotinylated Poly(ε-caprolactone)-block-poly(ethylene glycol)-block-poly(ε-caprolactone) (PCL-b-PEG-
b-PCL) Triblock Copolymers Based on Click Chemistry and Ring-Opening Polymerization
terminal alkynes was used as a strategy for preparation of PCL-b-
PEG-b-PCL triblock copolymers with biotin groups at the junction
points (Scheme 1). Alkynes functionalized PCL-b-PEG-b-PCL
triblock copolymers were synthesized by using alkynes functiona-
lized dihydroxyl PEG as macroinitiators for ROP of ε-caprolactone,
and click chemistry was employed in the synthesis of biotinylated
PCL-b-PEG-b-PCL triblock copolymers.
Scheme 2. Click Coupling Reaction of 3-Azidopropanol and
Tripropargylamine
’ EXPERIMENTAL SECTION
Materials. N-Hydroxysuccinimide (NHS) (Fluka, 97%) was purified by
recrystallization from a mixture of ethyl acetate and ethanol and dried under
vacuum. ε-Caprolactone (Shanghai Hao Sheng Trade Co., 99%) was distilled
before use. CuBr (99.5%) was purchased from Guo Yao Chemical Co. and
was purified by washing with glacial acetic acid and dried in a vacuum oven at
100 °C. L-Ascorbic acid sodium salt (Alfa, 99%), 3-bromo-1-propanol (Alfa,
99%), sodium azide (Alfa, 99%), 3-chloropropylamine hydrocholoride (Alfa,
Preparation of 3-Azidopropanol. The synthesis of 3-azidopropanol
was similar to previous literature.31 Sodium azide (1.92 g, 30.0 mmol)
and 3-bromo-1-propanol (2.5 g, 18 mmol) were added to a mixture of
acetone (30 mL) and water (5 mL). The mixture was stirred at refluxed
for 24 h. Acetone was removed on a rotary evaporator. The product was
extracted with ether, dried over anhydrous magnesium sulfate (MgSO4), and
filtered, and the solvent was removed by evaporation. The product was dried
under vacuum, and colorless oil was obtained (75% yield). 1H NMR (400
MHz) in CDCl3 (δ, ppm): 3.76 (t, 2H), 3.46 (t, 2H), and 1.84 (m, 2H).
Synthesis of 3-(4-((Dipropargylamino)methyl)-1,2,3-triazolyl)-
propanol (DPAMTP). DPAMTP was synthesized by click reaction
between 3-azidopropanol and tripropargylamine. The chemical reaction is
shown in Scheme 2. The synthesis is described as follows. 3-Azidopropanol
(0.10 g, 0.99 mmol) was dissolved in THF/water mixture (14 mL, 1:1, v/v),
97%), biotin (Sigma), CuSO4 5H2O (Tianjin Rui Jin Chemical Co., 99%),
3
EDC HCl (Shanghai Medpep Co., Ltd., 99%), PEG with number-average
3
molecular weight of 2000 g/mol (Tianjin Tian Tai Chemical Co., 98%),
methanesulfonyl chloride (Tianjin Guang Fu Chemical Co., 99%), tripro-
pargylamine (Aldrich, 98%), tin(II) 2-ethylhexanoate (Sn(Oct)2, Aldrich,
95%), avidin/2-(4-hydroxyphenylazo)benzoic acid (HABA) reagent
(Aldrich), and N,N,N0,N00,N00-pentamethyldiethylenetriamine (PMDETA,
Aldrich, 99%) were all used as received. All the solvents were distilled
before use.
and tripropargylamine (0.220 mL, 1.55 mmol), CuSO4 5H2O (8.0 mg,
3
0.032 mmol), and sodium ascorbate (80.0 mg, 0.404 mmol) were added into
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Scheme 3. Synthesis of Azido-biotin
Scheme 4. Synthesis of Azido-Terminated PEG (N3-PEG-
N3)
The synthesis of biotin-N3 is described as follows. To a solution of biotin-
NHS (0.14 g, 0.41 mmol) and triethylamine (0.12 mL, 0.86 mmol) in dry
DMF (16 mL) was slowly added 3-azidopropylamine (80 mg, 0.80 mmol).
After 24 h stirring at room temperature, the reaction solution was concen-
trated under reduced pressure, and a solid product was obtained. After
washing with water, the crude product was purified by gel column chor-
omatography (MeOH:CHCl3:AcOH = 8:1:0.05) to give the desired biotin-
N3 (100 mg, 77% yield). 1H NMR (400 MHz) in DMSO-d6 (δ, ppm): 7.84
(t, 1H), 6.42, 6.36 (d, 2H), 4.30, 4.13 (m, 2H), 3.33 (t, 2H), 3.10 (m, 3H),
2.86 (d, 1H), 2.60 (d, 1H), 2.06 (q, 2H), 1.69-1.20 (m, 8H).
Synthesis of Azido-Terminated PEG (N3-PEG-N3). Azido-
terminated PEG was synthesized by using a procedure similar to previous
literature.21 A typical procedure for the synthesis of azido-terminated PEG is
briefly described as follows (Scheme 4). In a dry round-bottom flask, HO-
PEG-OH (10 g, 5.0 mmol) with number-average molecular weight of 2000
was dissolved in 60 mL of toluene, and triethylamine (7 mL, 0.05 mol) was
added into the solution. The solution was then cooled to 0 °C, methane-
sulfonyl chloride (3.0 mL, 0.038 mol) was added dropwise, and the solution
was stirred at room temperature for 24 h. The solvent was removed under
reduced pressure. The solid was then dissolved in CH2Cl2 and washed
successfully with 1 M HCl solution, 1 M NaOH solution, and 1 M NaCl
solution. The organic layer was dried over anhydrous MgSO4 overnight,
precipitated in cold ether. After filtration, the yield mesylate-terminated
polymer (CH3O2SO-PEG-OSO2CH3) was dried under vacuum. Part of the
mesylate-terminated PEG (3.0 g, 0.0015 mol) was dissolved in 30 mL of dry
DMF; sodium azide (0.48 g, 0.0075 mol) was then added. The reaction
mixture was stirred magnetically at 50 °C for 24 h. After reaction DMF was
removed by rotary evaporation. The solid was dissolved in methylene
chloride, and the undissolved solid was removed by filtration. Before dried
over anhydrous MgSO4 overnight, the organic solution was washed twice by
water. After concentration of the organic solution on a rotatory evaporator,
azido-terminated PEG was precipitated in cold ether. After filtration and
drying under vacuum, dry polymer was obtained. 1H NMR (400 MHz) in
CDCl3 (δ, ppm), CH3O2SO-PEG-OSO2CH3: 3.08 (s, 6H), 3.43-3.84 (m,
172H), 4.37 (t, 4H). N3-PEG-N3: 3.42-3.84 (m, 172H), 3.37 (t, 4H).
Click Reaction between Azido-Terminated PEG and
DPAMTP. PEG macroinitiator with one hydroxyl group and one alkyne
group at each end, assigned as HA-PEG in this research, was synthesized by
“click” reaction between azido-terminated PEG and DPAMTP. A typical
reaction is described as follows. N3-PEG-N3 (0.50 g, 0.25 mmol) and
DPAMTP (1.16 g, 5.0 mmol) were dissolved in 4 mL of dry DMF in a
25 mL Schlenk flask, and PMDETA (53 μL, 0.25 mmol) was added. After
the mixture was bubbled with nitrogen for 15 min, CuBr (36 mg, 0.25 mmol)
the solution. The yellow solution was stirred for 24 h at room temperature.
The reaction mixture was evaporated under reduced pressure. The crude
product was dissolved in CH2Cl2 and washed with water until pH of the
solution reached 7.0. The organic layer was collected and dried with
anhydrous MgSO4. After filtration, the solvent was removed by rotary
evaporation, and the product was dried under vacuum to yield yellow oil.
The product was further purified by silica gel column chromatography with
a mixture of petro ether/ethyl acetate (1:1, v/v) as the eluent (33% yield).
1H NMR (400 MHz) in CDCl3 (δ, ppm): 7.65 (s, 1H), 4.52 (t, 2H), 3.92
(s, 2H), 3.65 (t, 2H), 3.50 (s, 4H), 2.30 (s, 2H) and 2.13 (m, 2H). MS
(chemical ionization): calcd for C12H17N4O: 233.287. Found: 233.35.
Synthesis of Azido-biotin (Biotin-N3). The synthesis of azido-
biotin is shown in Scheme 3. 3-Azidopropylamine was synthesized according
to previous literature.32 To a solution of 3-chloropropylamine hydrocholoride
(4.00 g, 0.0307 mol) in water (28 mL) at room temperature were added
NaN3 (10.0 g, 0.154 mol) and a pinch of KI. The reaction mixture was stirred
at 90 °C for 72 h. The reaction solution was cooled to room temperature, and
NaOH was added until pH reached 11. Solid NaCl was then added, and the
mixture was extracted five times with toluene. The combined organic layers
were dried with anhydrous MgSO4. After filtration, toluene was partially
removed under reduced pressure on a rotary evaporator at a low temperature.
On the basis of 1H NMR result, the concentration of the 3-azidopropylamine
in the resulting toluene solution was about 2.4 mmol/mL.
In order to synthesize biotin-NHS, EDC (245 mg, 1.28 mmol) was added
to a mixture of biotin (0.293 g, 1.20 mmol) and NHS (151 mg, 1.32 mmol)
in 22 mL of dry N,N-dimethylformamide (DMF).33 After 24 h stirring at
room temperature, the reaction solution was concentrated to yield a white
solid. The product was washed by methanol and dried under vacuum, and a
white solid was obtained with 69% yield. 1H NMR (400 MHz) in DMSO-d6
(δ, ppm): 6.42, 6.36 (d, 2H), 4.30, 4.15 (m, 2H), 3.11 (m, 1H), 2.86 (d,
1H), 2.82 (s, 4H), 2.67 (t, 2H), 2.60 (d, 1H), 1.71-1.34 (m, 6H).
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was added. The solution was degassed by three freeze-pump-thaw cycles.
The coupling reaction was carried out at 35 °C for 48 h and stopped by
exposure of the solution to air. Copper ions were removed from the polymer
solution with neutral alumina column chromatography. The product was
isolated by precipitation into cold diethyl ether. After filtration, the polymer
was dried under vacuum.
Malvern Instruments equipped with a 10 mW HeNe laser at a wavelength
of 633 nm. The results were analyzed in CONTIN mode. Mass spectrometry
was performed on a Finnigan LCQ advantage ion-trap mass spectrometer.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
(MALDI-TOF MS) was performed on a Bruker Autoflex III spectrometer
equipped with a 337 nm nitrogen laser. R-Cyano-4-hydroxycinnamic acid
was used as the matrix. Mass spectra were acquired in reflex mode at an
acceleration voltage of þ20 kV.
Synthesis of PCL-b-PEG-b-PCL Triblock Copolymers by
Ring-Opening Polymerization. HA-PEG was used as a macroinitia-
tor in ROP of CL. The chain length of PCL in the triblock copolymers was
controlled by controlling the monomer/macroinitiator ratio or monomer
conversion. A typical polymerization is described as follows. HA-PEG
(30 mg, 0.015 mmol) and ε-caprolactone (0.30 mL, 2.8 mmol) were
dissolved in 1 mL of dry DMF in a 10 mL Schlenk flask, and the mixture was
bubbled with nitrogen for 15 min. After 15 min stirring, Sn(Oct)2 (5.0 mg,
0.012 mmol) was added, and the solution was degassed by three freeze-
pump-thaw cycles. The polymerization was conducted at 110 °C for 24 h
and stopped by exposure to air. The polymer solution was precipitated in
cold methanol. After filtration, the polymer was dried under vacuum.
Synthesis of PCL-b-PEG-b-PCL Triblock Copolymers with
Biotins at the Junction Points. Biotin-N3 (11.7 mg, 0.0360 mmol)
and PCL-b-PEG-b-PCL (67.1 mg) were mixed in 0.5 mL of dry DMF in
a 10 mL Schlenk flask; PMDETA (63 μL, 0.30 mmol) and CuBr (44 mg,
0.30 mmol) were added after the mixture was bubbled with nitrogen for
15 min. The solution was degassed by three freeze-pump-thaw cycles.
The reaction was conducted at 80 °C for 24 h and stopped by exposure
to air. The polymer solution was dialyzed against aqueous phosphate
buffer solution (PBS, pH = 8.0, 0.05 M) and doubly distilled water to
remove excess biotin-N3 and other small molecules. The biotinylated
triblock copolymers were obtained after freeze-drying.
’ RESULTS AND DISCUSSION
As shown in Scheme 1, three steps are involved in the synthesis of
biotinylated triblock copolymers. At the first step, HA-PEG with one
hydroxyl and one alkyne group at each end was synthesized by
coupling reaction of azide-terminated PEG and DPAMTP. At the
second step, hydroxyl groups at the ends of HA-PEG chains were
used to initiate ROP of ε-caprolactone, and amphiphilic PCL-b-
PEG-b-PCL triblock copolymers with alkyne groups at the junction
points were prepared. On one block copolymer chain there are two
alkyne groups at the junction points. At the last step, biotin groups
were grafted to the junction points of PCL-b-PEG-b-PCL triblock
copolymer chains via click reaction.
3-Azidopropanol was prepared by reaction of 3-bromo-1-propanol
and NaN3 in dry DMF, and DPAMTP was synthesized by click
reaction between 3-azidopropanol and tripropargylamine
1
(Scheme 2). H NMR spectra of 3-azidopropanol and DPAMTP
are shown in Figure S1 (Supporting Information). The signals of
3-azidopropanol at 3.76, 3.46, and 1.84 ppm represent the methylene
protons next to the hydroxyl group (peak a), the methylene protons
next to the azide group (peak b), and the middle methylene protons
(peak c), respectively (spectrum a in Figure S1). After click reaction
with tripropargylamine, DPAMTP was obtained. The signals of
DPAMTP (spectrum b in Figure S1) at 7.65 ppm representing the
proton on the 1,2,3-triazole ring (g), at 4.52 ppm representing the
methylene protons next to the ring (b0), at 3.92 ppm representing the
methylene protons between the nitrogen atom and the 1,2,3-triazole
ring (e), at 3.50 ppm representing the methylene protons next to the
alkyne groups (f), and at 2.30 ppm representing the protons on alkyne
groups (peak d) are observed. The integral ratio of g:b0:e:f:d is
1:2:2:4:2, which indicates the successful synthesis of DPAMTP. The
molecular mass of DPAMTP measured by MS (MHþ, chemical
ionization) is 233.35, which is very close to the theoretic value at
233.28.
Avidin-HABA Assay Studies. The amount of available biotin on
micelles was determined by avidin/HABA reagent. The avidin/HABA
reagent was reconstituted with 10 mL of deionized water. In a 1 mL cuvette,
900 μL of HABA/avidin reagent was pipetted, and the absorbance was
measured at λ = 500 nm (AH50A0BA/Avidin) by a UV-vis spectrophotometer.
To this solution, 100 μL of sample was added, the solution was mixed by
inversion, and the absorbance at λ = 500 nm (AH50A0BA/AvidinþSample) was
read. The amount of the available biotin was calculated by the following
formula:34,35 μmol biotin/mL = (ΔA500/34) ꢀ 10, which corresponds to
the micromoles of biotin per milliliter of the sample solution, where ΔA500
HABA/AvidinþSample
= 0.9(AH50A0BA/Avidin) - A
.
500
Preparation of Polymer Micelles. Biotinylated PCL-b-PEG-b-
PCL triblock copolymer was dissolved in THF, and the solution was
added dropwise to 7-fold of doubly distilled water forming a cloudy
solution. After stirring for 1 day, the cloudy micellar solution was
transferred to a dialysis tubing (MWCO 7 kDa) and dialyzed against
doubly distilled water for 2 days.
N3-PEG-N3 was synthesized by a reaction of HO-PEG-OH with
methanesulfonyl chloride and followed by transformation of mesylate
end groups to azido groups via a reaction with NaN3 in dry DMF
(Scheme4). 1HNMRwasusedtocharacterize chemical structures of
Characterization. 1H NMR spectra were recorded on a Varian 400
spectrometer. Biotin-NHS and biotin-N3 were measured in deuterated
DMSO, and other products were measured in deuterated chloroform.
The apparent molecular weight and molecular weight distributions of the
PCL-b-PEG-b-PCL triblock copolymers were determined with a gel per-
meation chromatograph (GPC) equipped with a Waters 717 autosampler, a
Waters 1525 HPLC pump, three waters UltraStyragel columns with 5K-
600 K, 500K-30K, and 100K-10K molecular ranges, and a Waters 2414
refractive index detector. THF was used as eluent at a flow rate of 1.0 mL/
min. Molecular weights were calibrated on PS standards. Fourier transform
infrared absorption spectra (FTIR) were obtained on a Bio-Rad FTS6000
system using diffuse reflectance sampling accessories. Transmission electron
microscopy (TEM) observations were carried out on a Tecnai G2 20
S-TWIN electron microscope equipped with a Model 794 CCD camera.
TEM specimens were prepared by dipping copper grids into the micellar
solution (about 0.25 mg/mL) and drying in air. UV-vis absorption spectra
were collected on a JASCO V-570 spectrometer. Dynamic light scattering
(DLS) measurements were conducted on a Zetasizer Nano ZS from
1
the obtained polymers. H NMR spectra of mesylate-terminated
(spectrum a) and azido-terminated PEG (spectrum b) are shown in
Figure S2 (Supporting Information). In the spectrum of mesylate-
terminated PEG, the peak at 3.08 ppm represents the methyl protons
at mesylate end groups, and the peak at 4.37 ppm represents the
methylene protons next to the ester groups. The integral ratio of peak
a to peak b is 3:2, which indicates that the esterification reaction is
quantitative. After a reaction with NaN3, the signals of mesylate-
terminated PEG at 3.08 and 4.37 ppm disappeared completely, and
a new peak at 3.37 ppm representing the methylene protons next to
the azido groups is observed in the 1H NMR spectrum of the azide
chain-end-modified PEG.
Linear HA-PEG with one hydroxyl group and one alkyne group
at each end was synthesized by click coupling reaction between N3-
PEG-N3 and DPAMTP. In multistep click reactions side products
may be produced.36 Two N3-PEG-N3 macromolecules may react
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Figure 2. 1H NMR spectrum of HA-PEG.
the high viscosity in the solution, and coupling reaction between a
polymer chain and a small molecule is favorable. So the yield of HA-
PEG is higher at a higher polymer concentration. Integration results
of these peaks provided quantitative information about the fraction
of different species. When the concentration of N3-PEG-N3 was 5 ꢀ
10-3, 2 ꢀ 10-2, and 4 ꢀ 10-2 g/mL, the fraction of HA-PEG was
0.65, 0.69, and 0.80, respectively.
The influence of the molar ratio of alkyne to azido groups on the
synthesis of HA-PEG was also investigated in this research. Keeping
the concentration of N3-PEG-N3 at 4 ꢀ 10-2 g/mL, HA-PEG was
prepared at different molar ratios of DPAMTP to azido groups.
Figure 1b shows the GPC chromatograms of polymers prepared at
different molar ratios. When the molar ratio was 1.1:1 or 1.3:1, three
peaks were observed on the GPC curves. However, when the molar
ratio reached 5.0:1, peak c disappeared. With further increase of the
molar ratio, the intensity of peak b became very smaller. The
fractions of HA-PEG in the products prepared at molar ratios of
5.0:1 and 10:1 were 0.94 and 0.96, respectively. High purity target
polymer was prepared at high molar ratio of DPAMTP to azido
groups. Excess DPAMTP was removed by repeated dissolving and
precipitation cycles using system of dichloromethane/diethyl ether
(solvent and precipitant for PEG). GPC curves proved that after
four dissolving and precipitation cycles excess DPAMTP was
removed completely (Figure S3 in Supporting Information).
Figure 2 shows the 1H NMR spectrum of HA-PEG. In Figure 2
peaks at 3.23-4.20 ppm represent the overlapped signals of oxy-
methylene protons (-OCH2CH2-) in PEG (i, δ = 3.65 ppm),
methylene protons -NCH2C- (e, δ = 3.92 ppm), -CCH2O- (a,
δ = 3.65 ppm), and -CCH2N- (f, δ = 3.50 ppm) in DPAMTP
structure. In the spectrum signals at 7.78 ppm (peak g) representing
the -CH- proton on the triazole ring, at 2.15 ppm (peak c)
representing the methylene protons at terminal ends, and at 2.30
ppm (peak d) representing the protons on alkyne groups were also
observed. The integral ratio of peak g to c to d is about 2:2:1, which
indicates there is one alkyne group and one hydroxyl group at each
end, and hence proves successful “click” reaction between DPAMTP
and N3-PEG-N3 (Scheme 1). MALDI-TOF MS of HA-PEG was
also measured to prove the target polymer structure. As shown in
Figure 3, there are two series of repeating peaks, both of which have an
interval of 44, representing the molecular weight of ethylene oxide
unit. The molecular formula of HA-PEG is 44n þ 274 ꢀ 2 þ 28,
Figure 1. (A) GPC elution chromatograms of PEG with one hydroxyl
group and one alkyne group at each end (HA-PEG) prepared at different
polymer concentrations. The molar ratio of 3-(4-((dipropargylamino)-
methyl)-1,2,3-triazolyl)propanol (DPAMTP) to azido groups is 1.3:1 in
these reactions. (B) GPC elution chromatograms of HA-PEG prepared at
different molar ratios of DPAMTP to azido groups. The concentration of
N3-PEG-N3 is 4 ꢀ 10-2 g/mL in these reactions.
with one DPAMTP, producing the coupling product. The PEG
coupled polymer may further react with DPAMTP and N3-PEG-
N3, and macromolecules with three PEG segments in a polymer
chain structure may be produced. Besides target product HA-PEG,
macromolecules with two or three PEG segments in a structure are
possibly produced. In order to investigate possible side reactions in
the click reactions and optimize reaction conditions, controlled
experiments were conducted. In the experiments, the concentration
of N3-PEG-N3 or the molar ratio of azido to alkyne was controlled
while the other reaction conditions were kept unchanged. Figure 1a
shows the GPC chromatograms of the products prepared at
different polymer concentrations. On each GPC curve, three peaks
can be observed. On the basis of Figure 1, it is reasonable to expect
that peak a with a peak molecular weight of 3.5K represents our
target product HA-PEG, peak b with a peak molecular weight of
6.5K represents a polymer with two PEG segments in a chain, and
peak c with a peak molecular weight of 9.9K represents a polymer
with three PEG segments in a chian. All the three GPC curves in
Figure 1a were normalized at retention time of 20.8 min (the
position of peak a). The intensities of peaks b and c decreased with
the increase of polymer concentration, indicating that there were
less side reactions at higher polymer concentrations. The produc-
tion of all the side products is related to the coupling reactions
between two polymer chains. At a high polymer concentration click
coupling reaction between two polymer chains is unfavorable due to
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Figure 4. GPC elution chromatograms of the azide chain-end-modified
PEG (N3-PEG-N3), HA-PEG, and PCL75-b-PEG-b-PCL75 triblock
copolymer.
Figure 3. Matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrum of HA-PEG. M = 44n þ 274 ꢀ 2 þ
28. Matrix: R-cyano-4-hydroxycinnamic acid; cation source: Hþ and
Naþ in glassware, solvents, and reagents.
where n is 40. The two series of repeating peaks are attributed to HA-
PEG plus Hþ and Naþ. The molecular mass for HA-PEG plus Hþ
and HA-PEG plus Naþ were calculated to be 2338.53 and 2360.53,
respectively, which agreed well with the results of MALDI-TOF MS
(2340.4 and 2362.4). This result confirmed the success of click
reaction between N3-PEG-N3 and DPAMTP and high purity of the
target product HA-PEG.
The hydroxyl groups at two ends of a HA-PEG chain were used to
initiate ROP of ε-caprolactone, and PCL-b-PEG-b-PCL triblock
copolymers with alkyne groups at the juntion points were prepared.
In this research a series of PCL-b-PEG-b-PCL triblock copolymers
were synthesized by using Sn(Oct)2 as catalyst, and the triblock
copolymers were assigned as PCLn-b-PEG-b-PCLn, where n is the
average polymerization degree of PCL blocks. GPC elution chroma-
tograms of N3-PEG-N3, HA-PEG, and PCL75-b-PEG-b-PCL75 are
shown in Figure 4. After ROP, the GPC curve of the triblock
copolymer moves to high molecular weight part. It is also noted that
the molecular weight distribution of the triblock copolymer (PDI =
1.40) is broader than the two precursors, HA-PEG and N3-PEG-N3.
The 1H NMR spectrum of PCL20-b-PEG-b-PCL20 triblock copoly-
mer is shown in Figure 5a. In the spectrum, the peaks at 4.06 ppm (f)
and 3.65 ppm (a) represent the methylene protons in PCL block and
oxymethylene protons -OCHH2CH2- in PEG block, respectively.
By using the integral ratio of the two peaks, the molar ratio of PCL to
PEG can be obtained. The number-average degree of polymerization
Figure 5. 1H NMR spectra of triblock copolymer with alkyne groups at
the junction points (a) and biotinylated triblock copolymer (b).
biotin-N3 was removed by dialysis in aqueous PBS solution, and the
biotin-functionalized triblock copolymers were lyophilized.
1H NMR spectrum of biotin-functionalized PCL20-b-PEG-b-
PCL20 triblock copolymers is shown in Figure 5b. After click
reaction, featured signals of biotin molecules were observed. In
1
the H NMR spectrum of biotinylated PCL20-b-PEG-b-PCL20
triblock copolymer, the signal at 4.56 ppm (g) represents -
NHCHCH2S- protons in biotin molecules, and the signal at
1.26 ppm (h) represents the methylene protons next to the biotin
rings (Figure 5b). Because the signal at 4.06 ppm (f0) represents the
methylene protons in PCL block and the signal at 3.65 ppm (a0)
represents oxymethylene protons in PEG block, the average number
of biotin molecules in a triblock copolymer chain can be calculated
based on the integral ratio of peak g to f0 to a0. Our calculation result
shows that the ratio is about 4:82:174, which is very close to the
theoretical value. This result proved the successful click reaction
between biotin-N3 and PCL-b-PEG-b-PCL triblock copolymers
with alkyne groups at the junction points.
1
obtained by H NMR, number-average molecular weights, and
molecular weight distributions of all the triblock copolymers mea-
sured by GPC are summarized in Table 1.
It is well-known that biotin binds with high affinity to avidin and
streptavidin. The avidin-biotin interaction is the strongest known
noncovalent biological recognition.37,38 To investigate the applica-
tion of the triblock copolymers in bioconjugation, PCL-b-PEG-b-
PCL triblock copolymers with alkyne groups at the junction points
were functionalized with biotin-N3 by “click” reaction to obtain
biotinylated PCL-b-PEG-b-PCL triblock copolymers. Scheme 3
illustrates the synthesis of biotin-N3. After click reaction excess
Previous researches indicate that triblock copolymers can
form micelles in solvents that preferentially dissolve the middle
block.39 For example, spherical micelles formed by BAB triblock
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Table 1. Summary of Molecular Weights and Molecular Weight Distributions of PCL-b-PEG-b-PCL Triblock Copolymers
b
samples
average repeating unit number of PCLa
number-average molecular weights (Mn ꢀ 10-3
)
polydispersityb
PCL20-b-PEG-b-PCL20
PCL50-b-PEG-b-PCL50
PCL75-b-PEG-b-PCL75
PCL130-b-PEG-b-PCL130
20
50
4.6
6.0
1.67
1.50
1.40
1.62
75
7.2
130
10.0
a Measured by 1H NMR. b Measured by GPC.
Figure 6. TEM images of self-assembly aggregates of biotinylated PCL20-b-PEG-b-PCL20 triblock copolymer in aqueous solution before (a) and after
addition of avidin (b) and schematic representations for the interaction of biotinylated triblock copolymer micelles with avidin (c).
copolymers in solvents that preferentially dissolve A block, would
consist of a core containing B blocks, surrounded by loops of A
blocks. The self-assembly of amphiphilic biotinylated PCL20-b-
PEG-b-PCL20 triblock copolymer in aqueous solution was
investigated in this research. The triblock copolymer was dissolved
in THF, and the THF solution was added dropwise to 7-fold of
double-distilled water under vigorous stirring forming a cloudy
solution. The solution was dialyzed against double-distilled water for
2 days to remove THF. TEM observations were conducted to study
the structure of the self-assembled aggregates. The TEM specimens
were stained in hydrazine atmosphere for 8 h and then in OsO4
atmosphere for 100 min, and thus PCL blocks together with biotin
molecules at the junction points were stained.29 Figure 6a shows a
TEM image of self-assembly of the triblock copolymer in aqueous
solution. The triblock copolymer chains self-assembled into micellar
structures in aqueous solution. The average size of the spherical dots
representing micellar cores is about 10 nm. According to DLS
results, the average hydrodynamic diameter of the micelles is about
35 nm (Figure 7). In aqueous solution, the collapsed hydrophobic
PCL blocks constitute the cores of the micelles to avoid unfavorable
interaction with water, while the hydrophilic PEG blocks form the
coronae to stabilize the structures. In a triblock copolymer chain, the
PEG block connects two PCL blocks at two ends. In order to bring
two hydrophobic blocks into the core of a micelle, the hydrophilic
PEG block must form a loop structure in the corona of a micelle.
The functional biotin groups at the junction points of the triblock
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The bioavailability of biotin at the interfaces of micelles to avidin
was evaluated by an avidin/HABA competitive binding assay. It is
known that the binding of HABA to avidin gives an absorption
maximum at 500 nm. However, when biotin or biotinylated micelles
are added, biotin molecules displace HABA from the complex with
avidin, and the absorbance at 500 nm decreases. The absorption
decrease is proportional to the concentration of biotin in the solution,
and the change in absorbance can be used to calculate the amount of
biotin. The assay was conducted by measuring the absorbance of the
avidin/HABA complex at 500 nm before and after mixing with the
biotinylated micellar solutions. Figure 8 shows UV-vis spectra of the
avidin-HABA complex before and after addition of biotinylated
PCL20-b-PEG-b-PCL20 triblock copolymer micellar solution. Upon
addition of the biotinylated micelles, the absorbance of the avidin/
HABA complex at 500 nm decreased from 0.746 to 0.627, indicating
that the biotin molecules in the polymeric micelles competitively bind
to avidin by replacing HABA molecules. The amount of available
biotin to avidin was calculated to be 12.7 nmol/mg polymer. It is
worthy of note that this value is much lower than 1H NMR result
(260 nmol/mg polymer). The lower accessibility of biotin molecules
to avidin can be explained by the fact that some biotin molecules at
the interfaces of micelles could not interact with avidin because of the
interface rigidity.29,30,34 Additionally, avidin has four biotin binding
sites, and it can bind biotin molecules on different polymeric micelles
inducing the formation of micelle aggregates, which would also
sterically prohibit interaction of biotin molecules with avidin.
Figure 7. Size distributions of self-assembly of biotinylated PCL20-b-
PEG-b-PCL20 triblock copolymer in aqueous solutions before (below)
and after addition of avidin (upper).
’ CONCLUSIONS
In summary, PCL-b-PEG-b-PCL triblock copolymers with biotin
groups at the junction points were synthesized by click chemistry
and ring-opening polymerization. The triblock copolymers can self-
assemble into micelles in aqueous solution. The hydrophobic PCL
blocks form the cores of micelles, the hydrophilic PEG blocks form
the coronae, and the biotin moieties distribute at the interfaces of
micelles. The avidin/HABA competitive binding assay proved the
bioavailability of the biotinylated micelles to avidin. Upon addition
of avidin molecules to the micellar solutions, the aggregation of
micelles was observed due to the interaction between biotin and
avidin. This type of polymeric micelles could find potential applica-
tions in the fields of targeted drug delivery, biodetection, etc. This
research provides a versatile method to prepare biodegradable block
copolymer bioconjugates.
Figure 8. UV spectra of avidin-HABA complex before and after
addition of biotinylated PCL-b-PEG-b-PCL triblock copolymer
micelles.
copolymers reside at the interface of the micelles. The structure is
shown in Figure 6c.
’ ASSOCIATED CONTENT
The interaction of biotin with avidin was proved by TEM. A
solution of biotinylated triblock copolymer micelles was incubated
with avidin (biotin/avidin = 4:1 molar ratio) for 10 h. On the TEM
image aggregation of micelles were observed (Figure 6b), which
indicated that biotin molecules on the different micelles binded to
the same avidin molecule, and the addition of avidin to the
biotinylated micelles induces the aggregation of micelles. A magni-
fied TEM image was shown in the inset of Figure 6b. The structure
of aggregated micelles was also shown in Figure 6c.
Supporting Information. 1H NMR, GPC elution chro-
S
b
matograms. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: nkliul@yahoo.com (L.L.); hyzhao@nankai.edu.cn (H.Z.).
The hydrodynamic diameters of biotinylated triblock copolymer
micelles before and after addition of avidin were measured by DLS.
From the size distribution curve shown in Figure 7, it can be seen that
before addition of avidin the block copolymer micelles possess a
unimodal size distribution with a hydrodynamic diameter range from
25 to 80 nm. After addition of avidin, the size distribution moves to
big size part with a size range from 70 to 220 nm. The change in size is
due to the binding of avidin to the biotin molecules at the interfaces
of micelles and the production of micellar aggregates.
Author Contributions
§Jie Jin and Dongxia Wu contributed equally to this work.
’ ACKNOWLEDGMENT
This project was supported by National Natural Science
Foundation of China (NSFC) under Contracts 21074058 and
20874050.
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