Lectin recognizing thermoresponsive double hydrophilic glycopolymer micelles by RAFT polymerization

Article abstract of DOI:10.1039/c4ra04874a

Thermoresponsive double hydrophilic block glycopolymer poly(N- isopropylacrylamide-co-6-O-vinyladipoyl-d-glucose)-b-poly(N-isopropylacrylamide) (P(NIPAm-co-OVAG)-b-PNIPAm) was prepared by a combination of enzymatic synthesis and reversible addition-fragment chain transfer (RAFT) polymerization protocols using P(NIPAm-co-OVAG) as a macro-RAFT agent. All the precisely synthesized glycopolymers were characterized by nuclear magnetic resonance spectroscopy and gel permeation chromatography. Detections using laser light scattering and transmission electron microscopy revealed that the block glycopolymer was able to self-assemble into micelles with various sizes and sphere morphologies in aqueous solutions. The glucose pendants in the glycopolymers had obvious interaction with lectin, concanavalin A (Con A), and their capacity of interaction with Con A was controlled by the number of glucose units in the glycopolymers. The block glycopolymer micelles have excellent biocompatibility with pig iliac endothelial cells using the MTT assay, however the glycopolymer-Con A micelles could be used to induce apoptosis in human hepatoma SMMC-7721 cells.

Full text of DOI:10.1039/c4ra04874a

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PAPER
Lectin recognizing thermoresponsive double
hydrophilic glycopolymer micelles by RAFT
polymerization
Cite this: RSC Adv., 2014, 4, 34912
a
b
a
a
a
*
*
Kan Sun, S. W. Annie Bligh, Hua-li Nie, Jing Quan and Li-min Zhu
Thermoresponsive double hydrophilic block glycopolymer poly(N-isopropylacrylamide-co-6-O-
vinyladipoyl-D-glucose)-b-poly(N-isopropylacrylamide) (P(NIPAm-co-OVAG)-b-PNIPAm) was prepared
by
a
combination of enzymatic synthesis and reversible addition-fragment chain transfer (RAFT)
macro-RAFT agent. All the precisely
polymerization protocols using P(NIPAm-co-OVAG) as
a
synthesized glycopolymers were characterized by nuclear magnetic resonance spectroscopy and gel
permeation chromatography. Detections using laser light scattering and transmission electron
microscopy revealed that the block glycopolymer was able to self-assemble into micelles with various
sizes and sphere morphologies in aqueous solutions. The glucose pendants in the glycopolymers had
obvious interaction with lectin, concanavalin A (Con A), and their capacity of interaction with Con A was
controlled by the number of glucose units in the glycopolymers. The block glycopolymer micelles have
excellent biocompatibility with pig iliac endothelial cells using the MTT assay, however the
glycopolymer–Con A micelles could be used to induce apoptosis in human hepatoma SMMC-7721 cells.
Received 23rd May 2014
Accepted 31st July 2014
DOI: 10.1039/c4ra04874a
www.rsc.org/advances
specicity for a-D-glucose and a-D-mannose, and can induce
cancer cell death.14–17 The protein recognition is closely related
Introduction
to the sugar moiety of the glycopolymer, for example, a glyco-
polymer containing galactose can recognize peanut agglutinin
(PNA).44
Functional double hydrophilic block copolymers (DHBCs)
containing at least one functional unit such as a temperature-
responsive, light-sensitive, and pH-sensitive monomer have
recently attracted signicant interest for potential applications
in drug delivery systems,1–4 switchable catalysts,5 and gene
delivery vectors.6 Thermoresponsive DHBCs are one of the most
important candidates of biomaterials in DHBCs and have
capability of reversible micellization and dissociation
responding to the change of temperature. When the tempera-
ture is above the lower critical solution temperature (LCST) of
the thermoresponsive copolymer, the DHBCs self-assemble into
micelles. Inversely, the formed micelles dissociate into aqueous
solution when the temperature is below the LCST. Many studies
have been focused on the connection of thermoresponsive
DHBC with other functional monomer such as sugar-functional
monomer, which are deemed to be promising for protein
binding.7,8
The architecture of glycopolymer plays a crucial role in its
interaction with proteins.18,19 The precisely synthesized glyco-
polymers with great potential for use as novel biomaterials can
be produced by controlled polymerization technique.18–20 RAFT
polymerization as one of the most useful and amenable poly-
merization techniques can easily generate polymeric systems
and control structures for a wide-variety of polymers with
dened end and pendant functionalities, controlled molecular
weights, narrow polydispersities and low toxicity using mild
conditions.21–35 Miura et al. reported the advantage of RAFT
polymerization was that it provided precise glycopolymer
characteristics such as molecular weights with narrow poly-
dispersities and random saccharide spacing.20
In this study we aimed to obtain a series of well-dened
thermoresponsive double hydrophilic block glycopolymers
which could be used to recognize protein in a controlled fashion
and are not toxic. Thermoresponsive glycopolymers were
The glycopolymers that contain sugar pendant groups have
attracted chemists to investigate for their biomedical applica-
tions and protein recognition.9–11 They have been used to mimic
biological systems such as binding lectins through multipoint
interactions.12,13 As a lectin, concanavalin A (Con A) shows high
fabricated through polymerization of
a sugar-functional
monomer 6-O-vinyladipoyl-D-glucose (OVAG) and a tempera-
ture sensitive monomer NIPAm via controlled RAFT polymeri-
zation using S-1-dodecyl-S0- (a,a0- dimethyl-a00-acetic acid)
trithiocarbonate (DDATC) as a chain transfer agent (CTA) and
2,20-azo-bis-iso-butyronitrile (AIBN) as an initiator. The synthesis
and characterization of well-dened thermoresponsive block
aCollege of Chemistry, Chemical Engineering and Biotechnology, Donghua University,
Shanghai, 201620, P.R. China. E-mail: lzhu@dhu.edu.cn; Tel: +86 21 67792748
bDepartment of Life Sciences, Faculty of Science and Technology, University of
Westminster, 115 New Cavendish Street, London W1W 6UW, UK
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glycopolymers poly(NIPAm-co-OVAG)-b-poly(NIPAm) by RAFT
polymerization using poly(NIPAm-co-OVAG) as a macroagent are
presented. The binding ability of these glycopolymers to Con A,
their thermoresponsiveness, cell biocompatibility in free and
bound lectin forms with pig idiliac endothelial (PIE) cells and
human hepatoma SMMC-7721 cells are also reported.
Chain extension polymerization of P(NIPAm-co-OVAG) with
NIPAm (Experiment 4 in Table 1). Puried P(NIPAm-co-OVAG)
from experiment 1 (0.30 g, 6.31 ꢀ 10ꢁ6 mol, Mn(GPC) ¼ 4.75 ꢀ
104 g molꢁ1, Mw/Mn ¼ 1.26) was measured into a 25 mL one-
neck round-bottom ask followed by addition of NIPAm (0.22
g, 1.95 ꢀ 10ꢁ3 mol). The mixture was dissolved in DMF (1 mL)
and degassed by three freeze–thaw cycles and sealed under
ꢂ
vacuum. The polymerization was carried out at 70 C for 8 h.
Experimental
Materials
The diblock copolymer was dialyzed in double distilled water
for 24 h to remove residual monomers, the dialyzed solution
was freeze-dried to yield a ne slightly yellowish powder.
N-Isopropylacrylamide (NIPAm) (98%, Tokyo Chemical Co. Ltd.)
was puried by recrystallization in n-hexane. Alkaline protease
from Bacillus subtilis (EC 3.4.21.14, a crude preparation of
alkaline serine protease, powder, 100 U per mg) was purchased
from the Wuxi Xue Mei Technological Co. Ltd. (Wuxi, PR China).
Dimethyl formamide (DMF) was purchased from the Sinopharm
Chemical Reagent Co. (Shanghai, PR China). 2,20-Azo-bis-iso-
butyronitrile (AIBN; Sinopharm Chemical Reagent Co., Ltd.,
Shanghai, PR China, 97%) was puried by recrystallization from
a 95% water solution. Adipic acid was purchased from the
Sinopharm Chemical Reagent Co. (Shanghai, PR China). S-1-
dodecyl-S0- (a, a0- dimethyl-a00-acetic acid) trithiocarbonate
(DDATC) was purchased from Sigma-Aldrich (Shanghai) Trading
Co., Ltd. 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic
acid (HEPES) was procured from the Nanjing Robiot Co. (Nanj-
ing, China). Concanavalin A (Con A) was obtained from Sigma-
Aldrich (Shanghai, China). Fluorescently-labeled Con A (FITC-
Con A) was purchased from Vector Laboratories (Burlingame,
CA, USA). Dimethyl sulfoxide (DMSO) was purchased from the
China National Medicines Corporation Ltd. (Beijing, China).
Dulbecco's modied Eagle medium (DMEM), penicillin–strep-
tomycin and trypsin–EDTA solutions were obtained from Gibco
(Tulsa, OK, USA). All solvents used in this work were of analytical
Analytical Techniques
Nuclear Magnetic Resonance (NMR) spectra. All 1H NMR
spectra were recorded by dissolving 10 mg of puried samples
in D2O and using a Bruker DRX 400 MHz spectrometer (Bruker,
Rheinstetten, Germany).
Gel Permeation Chromatography (GPC). The molecular
weights (Mw and Mn) and molecular weight distributions of
glycopolymers were determined by GPC measurements on a
Waters LS measurement system using THF as the eluent, at a
ow rate of 1.0 mL minꢁ1 with a column temperature of 35 ꢂC.
The molecular weights (Mw and Mn) and molecular weight
distributions (Mw/Mn) for the sample polymers were calibrated
with standard polystyrene samples.
Critical Micelle Concentration (CMC) measurements of gly-
copolymers. The CMC of P(NIPAm-co-OVAG)-1 and P(NIPAm-co-
OVAG)-b-PNIPAm-1 were characterized by the uorescence
spectra (QM/TM, PTI, USA) and pyrene as the uorescence
probe. Pyrene solutions (10 mL, 6.576 ꢀ 10ꢁ4 M in acetone) were
added to the open tubes, and the acetone was evaporated
overnight. 10 mL of glycopolymer solutions at different
concentrations (2.0, 1.0, 0.5, 0.1, 0.01 and 0.001 mg mLꢁ1) were
added to tubes containing evaporated pyrene. These solutions
were stored at room temperature for 24 h to reach solubilization
equilibrium. Fluorescence emission spectra were recorded from
350 to 500 nm with an excitation wavelength of 334 nm and a
silt-width of 2 nm. The intensity ratio of I384/I374 from the
˚
grade and were dried by storing over activated 4 A molecular
sieves for 24 h prior to use. All other reagents were used as
received. Water was distilled before use.
Syntheses
Synthesis of 6-O-vinyladipoyl-D-glucose (OVAG). OVAG were emission spectra was analyzed to obtain CMC of P(NIPAm-co-
synthesized via the chemoenzymatic synthesis of carbohydrates OVAG)-b-PNIPAm-1 and P(NIPAm-co-OVAG)-1.
as described in the literature.36,37
Lower Critical Solution Temperature (LCST) determination
RAFT polymerization of OVAG and NIPAm (Experiment 1 in of glycopolymers. The thermoresponsive abilities of glycopol-
Table 1). In a typical experiment, NIPAm (0.79 g, 7.00 ꢀ 10ꢁ3 ymers were investigated by UV-vis spectrophotometer (Lambda
mol) was rst weighed in a 25 mL one-neck round-bottom ask, 35, Perkin Elmer, USA). A thermostatically controlled cuvette
and 25 mol% of OVAG (0.58 g, 1.75 ꢀ 10ꢁ3 mol) based on the was used, and the heating rate was 0.5 C minꢁ1. Absorbance
ꢂ
amount of NIPAm used was introduced into the same ask. N,N- through aqueous solutions of glycopolymer (1.0 mg mLꢁ1) at
Dimethylformamide (DMF, 1.50 mL) was later used to dissolve 500 nm was recorded at different temperatures. The LCST was
the monomers in the ask. S-1-dodecyl-S0-(a,a0-dimethyl-a00- dened as the temperature corresponding to a 50% of the
acetic acid) trithiocarbonate (DDATC, 3.15 ꢀ 10ꢁ2 g, 8.75 ꢀ maximum absorbance.37
10ꢁ5 mol) was nally added to the solution as chain transfer
Laser Light Scattering (LLS) measurement of block glyco-
agent (CTA). The mixture was then degassed by three freeze– polymer. P(NIPAm-co-OVAG)-b-PNIPAm-1 was dissolved in
thaw cycles and sealed under vacuum. The polymerization was double distilled water to prepare an aqueous solution with
carried out at 70 ꢂC for 12 h. The product was dialyzed in double glycopolymer concentration of 0.8 mg mLꢁ1. The sample was
distilled water for 24 h to remove residual monomers, the dia- ltered through a 0.45 mm Millipore lter into a clean scintil-
lyzed solution was freeze-dried into slightly yellowish solids lation vial. Laser light scattering (LLS) system (BI-200SM,
with a yield of 63%.
Brookhaven, USA), combined static laser scattering (SLS) and
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dynamic laser scattering (DLS), was used at l ¼ 532 nm. To passaging, the cells were trypsinized with 0.5/0.2% (v/v)
obtain average hydrodynamic radius (Rh) and average gyration trypsin–EDTA solution. Human hepatoma SMMC-7721 cells
radius (Rg) by LLS at different temperature, the ltered sample were cultured in a 1640 medium supplemented with 10% fetal
was heated at different temperatures for 15 min.
bovine serum, and 1% (v/v) penicillin–streptomycin solution,
Transmission Electron Microscopy (TEM). The TEM micro- and the culturing and passaging procedures are the same as
graphs were obtained by using a transmission electron micro- above.
scope (JEM-2100, JEOL, Japan). The samples were prepared by
MTT assays. The cytotoxicity of the RAFT-based glycopoly-
casting an aqueous solution of glycopolymer micelle solution mers P(NIPAm-co-OVAG)-b-PNIPAm-1 and P(NIPAm-co-OVAG)-1
(0.4 mg mLꢁ1) onto a copper grid with thin lms of Formvar and was evaluated by MTT assay. PIE cells were seeded into 96-well
carbon successively at 38 ꢂC. The images taken were also used to plates at a density of 20 000 cells per well and incubated for 24
P
obtain an average particle size using the equation Dn ¼ niDi/ h. The glycopolymers in PBS solutions were added into the cells
P
ni. Thirty particles were each measured from the images and (nal concentrations were 1, 5, 10, 25, 50, 100 mg Lꢁ1). Aer
calculated using the equation, where Dn is the number-average incubation for another 24 h, 20 mL of MTT solution (5 mg mLꢁ1
diameter.
in PBS buffer) was added to each well. Four hours later, the
medium was removed and the samples in the wells were air
dried. 200 mL of DMSO was added to dissolve the formed crys-
tals. The optical density of the solution was measured at 570 nm
using a microplate reader (Thermo Multiskan MK3, Thermo
Scientic Company, Waltham, UK). The PIE cells without any
treatment were used as the control.
Lectin-binding assay
Turbidity. The lectin agglutination activity of the glycopol-
ymers micelles was evaluated by measuring the change in the
turbidity of buffer solution because of the multipoint inter-
action between the glycopolymer and Con A. Buffer solution
(pH ¼ 7.4) for lectin-binding assay was prepared by adding 10
mM HEPES, 0.15 M NaCl, 0.1 mM CaCl2, 0.01 mM MnCl2.
The nal mixed solutions contained 7 mg mLꢁ1 glycopolymer
and 2 mg mLꢁ1 Con A. The turbidity was measured by using a
UV-vis spectrophotometer (Lambda 35, Perkin Elmer, USA) at
a wave-length of 600 nm. The interaction tests were repeated
at least three-times to ensure the measurement
reproducibility.
Temperature inuence on lectin-binding. The glycopolymers
P(NIPAm-co-OVAG)-b-PNIPAm-1 and P(NIPAm-co-OVAG)-1 were
dissolved in HEPES buffer solution (pH ¼ 7.4) to prepare solu-
tions with concentration of 0.4 mg mLꢁ1. The samples were
ltered through at a 0.45 mm Millipore lter into clean scintil-
lation vials, and were heated at 25 C and 37 C for 15 min to
measure the Dh. The Con A solution was added to glycopolymer
solutions to form the nal mixed solution containing 0.35 mg
mLꢁ1 glycopolymer and 0.15 mg mLꢁ1 Con A. Aer 1 h, the
mixed solution was heated at 25 ꢂC and 37 ꢂC for 15 min to
measure the Dh.
Confocal microscopy. Fluorescein isothiocyanate Con A
(FITC-Con A) was utilized to determine the protein recognition
with glycopolymer micelles, and a confocal laser scanning
microscope (Carl Zeiss LSM700, Jena, Germany) with four
channels (405, 488, 555, 634 nm) excitation was used. Glyco-
polymer and FITC-Con A all were dissolved in HEPES buffer
solution (pH ¼ 7.4). Before the determination of confocal, the
same volume of glycopolymers (7 mg mLꢁ1) and FITC-Con A (0.5
Further experiments of the cytotoxicity of glycopolymers
binding Con A were performed in which a series of solutions
with 1 g Lꢁ1 of P(NIPAm-co-OVAG)-b-PNIPAm-1 and different
concentration of Con A (0, 100, 300, 500, 1000 mg Lꢁ1) in HEPES
buffer were initially incubated for 4 h. SMMC-7721 cells were
seeded into 96-well plates at a density of 20 000 cells per well
and incubated for 24 h. The glycopolymers binding Con A in
HEPES buffer were added to the cells (nal concentrations of
Con A were 0, 10, 30, 50, 100 mg Lꢁ1 and glycopolymer was 100
mg Lꢁ1 in every well). They were then assayed as above. The
SMMC-7721 cells without any treatment were used as the
control.
ꢂ
ꢂ
Results and discussion
Polymer synthesis
Thermoresponsive block glycopolymers P(NIPAm-co-OVAG)-b-
PNIPAm with glucose was facilely synthesized by combining
enzymatic synthesis with RAFT polymerization (Scheme 1) with
good yields (Table 1). Diblock glycopolymers P(NIPAm-co-
OVAG)-b-PNIPAm werꢂe prepared by RAFT polymerization of
NIPAm in DMF at 70 C using P(NIPAm-co-OVAG) as a marco-
RAFT agent, which was prepared by RAFT polymerization of
NIPAm and OVAG with different feed molar ratios (Table 1). In
the polymerization reaction, S-1-dodecyl-S0-(a,a0-dimethyl-a00-
acetic acid)trithiocarbonate (DDATC) was used as a RAFT agent
because of its ability to control the reaction and its low toxicity
property.40 The molar ratio of monomers to DDATC was xed at
100 : 1. The conditions of the polymerization were listed in
Table 1.
mg mLꢁ1) were mixed keeping at 25 C for 2 h.
ꢂ
The1H NMR spectra of glycopolymers are shown in Fig. 1a
Cell viability study
Cell culture. Pig iliac endothelial (PIE) cells were main- and b, which reveal the presence of NIPAm and glucose
1
tained as an exponentially growing monolayer in a DMEM moieties and the absence of vinyl groups. H NMR of NIPAm
high sugar medium supplemented with 10% fetal bovine (D2O, 400 MHz) d (ppm): 6.02 (qd, 2H, 1-H), 5.56 (dd, 1H, 2-H),
serum, and 1% (v/v) penicillin–streptomycin solution. The 3.82 (m, 1H, 4-H), 1.00 (d, 6H, 5, 50-H). 1H NMR of OVAG (D2O,
ꢂ
cells were cultured at 37 C in a humidied atmosphere con- 400 MHz) d (ppm): 7.15 (dd, 1H, 13-H), 5.25 (d, 0.46H, 1-Ha),
taining 5% CO2. Cells were passaged every 1–2 days. For 4.85 (dd, 1H, 14-H trans), 4.65 (dd, 1H, 14-H cis), 4.55
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Scheme 1 (a) The synthesis of 6-O-vinyladipoyl-D-glucopyranose (OVAG) by controllable chemoenzymatic reaction; (b) the synthesis of
P(NIPAm-co-OVAG)-b-PNIPAm by RAFT polymerization.
Table 1 RAFT polymerization of glycopolymers
Polymera
[NIPAm]0/[OVAG]0/[CTA]0/[AIBN]0
Yieldd (%)
b,c
Experiment
1
2
3
4
5
6
PNO-1
PNO-2
PNO-3
PNO-b-PN-1
PNO-b-PN-2
PNO-b-PN-3
80/20/1/0.05
90/10/1/0.05
93/7/1/0.05
300/0/1/0.25
300/0/1/0.25
300/0/1/0.25
63
67
75
71
76
80
a
PNO and PNO-b-PN were respectively shorted for P(NIPAm-co-OVAG) and P(NIPAm-co-OVAG)-b-PNIPAm. b [NIPAm]0, [OVAG]0, [AIBN]0 and [CTA]0
represent the initial concentrations of monomers, initiator and chain transfer agents (CTA), respectively. c In the synthesis of P(NIPAm-co-OVAG),
CTA was S-1-dodecyl-S0-(a,a0- dimethyl-a00-acetic acid)trithiocarbonate (DDATC), and P(NIPAm-co-OVAG) was macroCTA in the polymerization of
P(NIPAm-co-OVAG)-b-PNIPAm. d Yield was determined by gravimetric analysis.
(d, 0.54H, 1-Hb), 4.25 (m, 2H, 6-H), 3.85 (m, 0.46H, 5-H), 3.60 calculated based on the integral values of the signals at d ¼
(m, 3-Ha), 3.55 (m, 5-Hb), 3.40 (m, 2-Hb), 3.35 (m, 3-Hb), 3.30 2.38–2.22 (2H of OVAG) and d ¼ 1.25–0.95 (6H of NIPAm) and
(m, 4-H), 3.15 (m, 0.52H, 2-Ha), 2.35 (m, 4H), 1.55(m, 4H). In the results are presented in Table 2. Meanwhile, when
1
the H NMR spectrum of P(NIPAm-co-OVAG)-b-PNIPAm-1, the compared with P(NIPAm-co-OVAG)-1, the characteristic signals
signals at d 7.15 (dd, 1H, 13-H) and 4.85 (dd, 1H, 14-H trans), of NIPAm are enhanced because of the additional PNIPAm
4.65 (dd, 1H, 14-H cis) of OVAG and 6.02 (qd, 2H), 5.56 (dd, block. The calculated monomer ratio are basically consistent
1H) of NIPAm have almost disappeared demonstrating poly- with the feed of the monomer ratio.
merization has proceeded. In addition, the molar ratio of
The molecular weights (Mn and Mw) and molecular weight
NIPAm and OVAG units in the resultant polymers was distributions (Mw/Mn) of glycopolymers measured by GPC in
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Fig. 1 1H NMR spectra of (a) P(NIPAm-co-OVAG)-b-PNIPAm; (b) P(NIPAm-co-OVAG); (c) OVAG; (d) NIPAm in D2O.
Table 2 Properties of glycopolymers prepared by RAFT polymerization
Polymera
Mn (ꢀ 104)
Mwb (ꢀ 104)
PDIb
[NIPAm]/[OVAG]c
LCSTd (ꢂC)
b
PNO-1
PNO-2
PNO-3
PNO-b-PN-1
PNO-b-PN-2
PNO-b-PN-3
4.75
4.55
5.36
7.42
7.26
7.79
6.00
5.49
6.72
9.17
8.41
9.27
1.26
1.20
1.25
1.23
1.16
1.19
5.9
9.1
15.6
9.7
13.8
20.3
38.0
37.0
36.0
36.5
35.5
33.0
a
b
PNO and PNO-b-PN were respectively shorted for P(NIPAm-co-OVAG) and P(NIPAm-co-OVAG)-b-PNIPAm. Mn, Mw and PDI were determined by
GPC. [NIPAm]/[OVAG] represented the molar ratio of NIPAm and OVAG in the resultant glycopolymers and was determined by 1H NMR.
c
d
LCST was determined by UV-vis spectroscopy.
THF are presented in Table 2. Compared to the macro-RAFT 9.17 ꢀ 104, Mw/Mn of P(NIPAm-co-OVAG)-b-PNIPAm-1 is 1.23.
agents, all the diblock glycopolymers eluted at higher molec- All the GPC traces of glycopolymers are symmetrical and
ular weights and a clear shi can be observed. Fig. 2 shows the unimodal, and the molecular weight distributions are relatively
Mn and Mw of P(NIPAm-co-OVAG)-b-PNIPAm-1 is 7.42 ꢀ 104 and narrow. Because of the polydisperse of polymer, the values on
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on the temperature of the glycopolymers micellar solution. The
LCST of diblock glycopolymer P(NIPAm-co-OVAG)-b-PNIPAm-1
at 1.0 mg mLꢁ1 was 36.5 C (Fig. 3). Overall, the LCST values
ꢂ
of glycopolymers increase with increasing molar fraction of
OVAG in the glycopolymers (Table 2).
Meanwhile, it can be found that phaseꢂ transition of
P(NIPAm-co-OVAG)-1 lies in the range of 35–41 C which is the
widest in the glycopolymers studied. It is due to the increase of
hydrogen bonds between hydroxyl groups of glucose and water
which retard the process of phase transition with the increasing
the molar ratio of OVAG. However, it also can be found that the
range of phase transition of P(NIPAm-co-OVAG)-b-PNIPAm-1 is
ꢂ
35–38 C and it is narrower than that of P(NIPAm-co-OVAG)-1.
The reason for this change is the increasing proportion of
PNIPAm and the formation of a block, which has also been
observed in other similar studies.42,43
Fig. 2 GPC traces of P(NIPAm-co-OVAG)-b-PNIPAm-1(red curve)
and P(NIPAm-co-OVAG)-1 (blue curve) in THF.
Self-assembled micellar behavior
Critical micelle concentration of glycopolymers. The critical
micelle concentration (CMC) of glycopolymers is derived from
steady-state uorescent probe studies. Pyrene has been widely
used as a uorescence probe in the polymeric micellar solu-
tion.38 As long as polymeric micelles are formed, pyrene was
preferentially distributed in the hydrophobic micelle cores,
meanwhile, the surrounding for pyrene changed from polar to
non-polar.37 In the uorescence emission spectra of pyrene, the
relative intensities of the rst and third peak (I1 at 374 nm and I3
at 384 nm) are useful in detecting the critical micelle concen-
tration (CMC) of polymeric micelles. The relative intensity ratio
(I3/I1) is a function of copolymer concentration which can be
used to determine the CMC of glycopolymers.
From the plot of the relative intensity ratio (I3/I1) versus gly-
copolymer concentration (Fig. 4), an abrupt increase in the
relative intensity ratio (I3/I1) was observed with increasing
Fig. 3 Turbidity curves recorded for 1.0 mg mLꢁ1 solution of glyco-
polymers in water on UV-vis spectroscopy at 500 nm and the heating
rate was 0.5 ꢂC minꢁ1
.
Mn and Mw (and thus, also of Mw/Mn) are the values of samples
with statistical means, which cannot be considered as the real
ones. Compared with the results by GPC, the Mn,NMR (molecular
weight determined by 1H NMR) is similar to the Mn,GPC, which is
shown in Table 2. Meanwhile, in order to reduce the error
caused by the structural difference between synthesized
samples and standard sample, universal calibration curve was
used. All data including Mn, Mw and Mw/Mn were the average
numbers of three replicates.
LCST determination of glycopolymers
Fig. 4 The critical micellar concentration (CMC) graphs of P(NIPAm-
co-OVAG)-b-PNIPAm-1 (blue graph) and P(NIPAm-co-OVAG)-1
(purple graph). A series of concentrations are 2, 1, 0.5, 0.1, 0.01 and
0.001 mg mLꢁ1. The CMC values of P(NIPAm-co-OVAG)-b-PNIPAm-1
and P(NIPAm-co-OVAG)-1 in aqueous media are about 0.19 g Lꢁ1 and
It has been reported that the LCST value of PNIPAm-based
copolymer can be precisely designed to be in the range 32–40
ꢂC by adjusting the relative amount of monomers, and LCST
value increases with the molar fraction of hydrophilic mono-
mers in the copolymers.37 Fig. 3 shows the absorbance depends 0.31 g Lꢁ1, respectively.
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Fig. 5 (a) The temperature dependences of average hydrodynamic
radius (Rh, blue square), and average gyration radius (Rg, red circle) of
P(NIPAm-co-OVAG)-b-PNIPAm-1 micelles in water at a concentra-
tion of 8 ꢀ 10ꢁ4 g mLꢁ1. (b) The temperature dependence of Rg/Rh for
P(NIPAm-co-OVAG)-b-PNIPAm-1 micelles in water.
Fig.
7
Turbidimetry assay experiments of P(NIPAm-co-OVAG)-1
(purple circle), P(NIPAm-co-OVAG)-2 (black square), P(NIPAm-co-
OVAG)-3 (red down triangle), P(NIPAm-co-OVAG)-b-PNIPAm-1 (blue
up triangle) micelles with Con A at 30 ꢂC.
than the LCST, the glycopolymer is molecularly soluble, Rh is
typically less than 35 nm and Rg is less than 65 nm. However, at
elevated temperature to 33–35 ꢂC, Rh and Rg increased
dramatically. This suggests the occurrence of the coil-to-globule
transition of the glycopolymer and aggregation of globose
nanoparticles is due to the increasing specic surface area. At
even higher temperatures, Rh and Rg maintain almost constant.
In order to understand in detail the process of the chain
conformation changes, the temperature dependence of Rg/Rh
were also studied (Fig. 5(b)). Rg/Rh is nearly a constant (ꢃ1.6) in
the heating process when temperature is lower than the LCST.
Water is a good solvent for the block polymer P(NIPAm-co-
OVAG)-b-PNIPAm-1, and glycopolymer is linear exible chain
coils in water. In the transition range of the heating process, Rg/
Rh decreases sharply from 1.6 to 0.7 and remains ꢃ0.7 when the
ꢂ
temperature is higher than 35 C. This indicates that PNIPAm
block and PNIPAm chain in the block polymer are induced from
the extended coil conformation to the collapsed compact
globule.
Transmission Electron Microscopy (TEM). Micelle
morphology of the block glycopolymers was investigated by
TEM (see Fig. 6), which shows the images observed from a
sample-loaded TEM grid. The number-average diameter (Dn) of
Fig. 6 TEM images of micelles self-assembled from P(NIPAm-co-
OVAG)-2 (a and b) and P(NIPAm-co-OVAG)-b-PNIPAm-1 (c and d) at
low (a and c) and high (b and d) magnification.
glycopolymer concentrations, indicating formation of micelles
and the transfer of pyrene into the hydrophobic core of the
micelles. The CMC values for P(NIPAm-co-OVAG)-b-PNIPAm-1
P(NIPAm-co-OVAG)-b-PNIPAm-1 is 12
ꢄ
3 nm, which is
measured from Fig. 6(c) and (d). It is evident that the block
glycopolymers were dispersed as self-assembled micelles with
uniform sizes and a regularly spherical shape. Compared to the
size by TEM, the average hydrodynamic diameter (Dh) of the
micelles by DLS is larger with the size at around 20 ꢄ 7 nm (see
Fig. 8). For P(NIPAm-co-OVAG)-1, the Dn is 25 ꢄ 5 nm (Fig. 6 (a)
and (b)) and the Dh is 32 ꢄ 10 nm (Fig. 8).
and P(NIPAm-co-OVAG)-1 were as low as 0.31 and 0.19 g Lꢁ1
,
respectively (Fig. 4), providing evidence for an apparent stability
of micelles, which is consistent with the literature.39
Thermoresponsive behavior of block glycopolymer micelles.
Thermoresponsive double hydrophilic block copolymer could
self-assemble into micelles and have been well characterized by
Rangelov et al.5,40 In order to investigate the thermoresponsive
phenomenon of diblock glycopolymer micellization, tempera-
ture dependences of both average hydrodynamic radius (Rh)
and average gyration radius (Rg) were investigated.
The reduction in size observed from the TEM study may be
due to the collapse of the free segment of the hydrophilic chain
of the polymer as well as the dehydration of the polymer chain.38
Lectin-binding assay
Fig. 5(a) shows the temperature dependence of Rh and Rg for
aqueous solution of P(NIPAm-co-OVAG)-b-PNIPAm-1. On heat-
Structure effect on lectin-binding. It is well known that gly-
copolymers with a large quantity of glucose units are very
attractive to serve as carriers for drug delivery or as systems for
ꢂ
ꢂ
ing, both Rh and Rg hardly changed between 25 C and 31 C.
Obviously, for the glycopolymer, when the temperature is lower
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was used to characterize the specic binding with glucose
moieties. The turbidity assay measures the changes in absor-
bance at a wavelength of 600 nm using UV-vis spectroscopy.
In Fig. 7, the turbidity increases with conjugation time to
reach plateaus aer 4 min and the maximum absorbance value
increases with the portion of OVAG in P(NIPAm-co-OVAG). It
proved that the more sugar moieties in glycopolymer and the
greater recognizability to Con A. Multivalent arrays displayed by
the glycopolymer allow high specic interactions between the
glucose ligands on the polymers. Meanwhile, the maximum
absorbance value of P(NIPAm-co-OVAG)-b-PNIPAm-1 is similar
to P(NIPAm-co-OVAG)-2 because of analogical molar ratio of
monomers. However, Fig. 7 also shows that glycopolymers
contain the same portion of OVAG and have the different rate of
increase of absorbance, P(NIPAm-co-OVAG)-b-PNIPAm-1 is a
little faster than P(NIPAm-co-OVAG)-2. The reason of this
phenomenon can be explained that the micellars of block gly-
copolymer may have more regular structure because of the
existence of double block.
Fig. 8 DLS experiments of the glycopolymers with Con A at different
temperature.
Temperature inuence on lectin-binding. In addition to the
turbidity test, dynamic light scattering measures were also
conducted to study the size growth (Fig. 8). The particle sizes
increased obviously when Con A was attached to the particles at
different temperature. Since Con A has four binding sides, inter-
micelles cross-linking occurred and led to increase of the
average hydrodynamic diameters. Fig. 8 shows that the average
hydrodynamic diameters (Dh) of P(NIPAm-co-OVAG)-2 increases
by about 110% aer the adding of Con A at 30 ꢂC and 53% at 37
ꢂC, and Dh of P(NIPAm-co-OVAG)-b-PNIPAm-1 increases by
about 95% aer the adding of Con A at 30 ꢂC and 50% at 37 ꢂC.
When the temperature is lower than the LCST of glycopolymer,
Fig. 9 Confocal microscopy images of interaction of glycopolymer
P(NIPAm-co-OVAG)-1 (a) and P(NIPAm-co-OVAG)-b-PNIPAm-1 (b)
with FITC-Con A.
ꢂ
the recognition is better than the temperature at 37 C (above
the LCST). Because random coil structure (under the LCST) of
glycopolymer micelle is more exible to bind lectin and more
sugar moieties are exposed to bind lectin.18 Besides, P(NIPAm-
co-OVAG)-2 is similar to P(NIPAm-co-OVAG)-b-PNIPAm-1 in
change of Dh at different temperature because of their similar
molar ratio of monomers.
purication because of their ability to protein recognition.41 In
order to prove that the bio-functionality of the glucose moieties
is still active and not lost during the RAFT polymerization
process and to understand how the protein-binding reaction
would proceed in the biological system, the lectin-binding
ability of the glycopolymer micelles was tested. Lectin Con A
Fig. 10 (a) Viability of PIECs after incubation with P(NIPAm-co-OVAG)-1 (purple bar) and P(NIPAm-co-OVAG)-b-PNIPAm-1 (blue bar) at various
concentrations; (b) viability of SMMC-7721 cells after incubation with P(NIPAm-co-OVAG)-b-PNIPAm-1 binding different concentrations Con A.
The viability was measured by a MTT assay.
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for Therapeutic Textiles, the State Key Laboratory for the
Modication of Chemical Fibers and Polymer Materials, the Key
Laboratory of Science &Technology of Eco-Textile, Ministry of
Education, and the Fundamental Research funds for the
Central Universities, Langsha Group, Jofo (WeiFang) Nonwoven
Co. Ltd.
Confocal microscopy. To be able to visualize the conjugation
of Con A in the glucose functionalized polymeric micelle and
conrm a successful conjugation, uorescein isothiocyanate
conjugated Con A (FITC-Con A) was employed. Fig. 9 shows the
interaction of glycopolymer with FITC-Con A. Fig. 9(a) depicts
P(NIPAm-co-OVAG)-1, in confocal mode, with green uores-
cence indicative of FITC-Con A. Fig. 9(b) depicts P(NIPAm-co-
OVAG)-b-PNIPAm-1, in confocal mode, with green uorescence
indicative of FITC-Con A.
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A series of multifunctional double hydrophilic block glycopoly-
mers in the form of self-assembling micelles were successfully
synthesized via controlled RAFT polymerization of a sugar-
functional vinyl ester monomer and a thermoresponsive mono-
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