Design of Electrophoretic and Biocompatible Poly(2-oxazoline)s Initiated by Perfluoroalkanesulfoneimides and Electrophoretic Deposition with Bioactive Glass

Article abstract of DOI:10.1021/acs.biomac.5b00043

N-Methyl bis[(nonafluorobutane)sulfonyl]imide (Nf₂NMe) was synthesized to serve as an initiator for polymerization of 2-oxazolines and the polymerization activity and control of molecular weight were compared with conventional methyl triflate (TfOMe). Ring-opening polymerization of the vinyl-containing 2-oxazoline, 2-(3-butenyl)-2-oxazoline, and a subsequent thiol-ene click reaction and oxidation resulted in the successful synthesis of electrophoretic poly(2-oxazoline)s containing pendent sulfone. It was possible to coat the polymeric sulfones obtained via oxone oxidation (conversion, >99%) on a stainless-steel anode selectively. Furthermore, hybridization of the poly(2-oxazoline)s with bioactive glass (Bioglass45S5) by electrophoretic deposition (EPD) was investigated, and the biocompatibility of the hybrid was also evaluated. (Figure Presented)

Full text of DOI:10.1021/acs.biomac.5b00043

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Article
Design of Electrophoretic and Biocompatible Poly(2-
oxazoline)s Initiated by Perfluoroalkanesulfoneimides
and Electrophoretic Deposition with Bioactive Glass
Terunari Hayashi, and Akinori Takasu
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00043 • Publication Date (Web): 04 Mar 2015
Downloaded from http://pubs.acs.org on March 11, 2015
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Biomacromolecules: Article(Revised)
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Design of Electrophoretic and Biocompatible Poly(2-oxazoline)s
Initiated by Perfluoroalkanesulfoneimides and Electrophoretic
Deposition with Bioactive Glass
Terunari Hayashi and Akinori Takasu*
Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of
Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
AUTHOR EMAIL ADDRESS: takasu.akinori@nitech.ac.jp
*To whom correspondence should be addressed: Telephone: +81-52-735-7159. Fax:
+81-52-735-5342. E-mail takasu.akinori@nitech.ac.jp
ABSTRACT
N-methyl bis[(nonafluorobutane)sulfonyl]imide (Nf2NMe) was synthesized to
serve as an initiator for polymerization of 2-oxazolines and the polymerization activity
and control of molecular weight were compared with conventional methyl triflate
(TfOMe). Ring-opening polymerization of the vinyl-containing 2-oxazoline,
2-(3-butenyl)-2-oxazoline, and a subsequent thiol-ene click reaction and oxidation
resulted in the successful synthesis of electrophoritic poly(2-oxazoline)s containing
pendent sulfone. It was possible to coat the polymeric sulfones obtained via oxone
oxidation (conversion, >99%) on a stainless-steel anode selectively. Furthermore,
hybridization of the poly(2-oxazoline)s with bioactive glass (Bioglass®45S5) by
electrophoretic deposition (EPD) was investigated, and the biocompatibility of the
hybrid was also evaluated.
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INTRODUCTION
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The phenomenon of electrophoresis was first observed in water in 1807.1 Since
then, many researchers have studied electrophoresis from a chemical physics
perspective. In 1933, electrophoretic deposition (EPD), a layer-forming technique, was
first used to deposit TiO2 and tungsten on a platinum cathode.2 The principle behind
EPD is that by applying a voltage to a dispersion, monodispersed particles can be
electrophoresed to layer deposited on a substrate. EPD can generate nano- to
micrometer thick films, even on complex substrate surfaces. In the field of biomedical
materials, new bioactive materials that can effectively coat a targeted metal substrate to
immobilize bioactive compounds including cells or enzymes for bioreactor and
biosensors and also to develop implant materials.3 However, there are a limited number
of reports dealing with inorganic/organic films formed using EPD.3a,3e,4
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2-Oxazolines are 5-membered cyclic imino ethers which were first synthesized
in 1889.5 The first polymerization of these compounds was reported by several
independent researchers in 1966-1967,6-8 with a cationic propagating center (cationic
ring-opening polymerization).9 The initiators of the polymerization are electrophiles
(e.g., Lewis acids,6-8 acid halides,10 methyl tosylate,9 and methyl triflate9). Using methyl
tosylate (TsOMe), methyl triflate (TfOMe), or an alkyl halide as an initiator under the
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appropriate conditions, 2-oxazolines undergo controlled-polymerization, affording
poly(2-oxazoline)s with well-defined structures. Poly(2-oxazoline)s have a variety of
characteristics,11, 12 including biocompatibility13 and thermoresponsiveness,14 and it is
possible to control the hydrophilic and hydrophobic balance by the length of pendent
substituents. Due to this facile procedure for the design of various structures,
poly(2-oxazoline)s are great candidates for new biomaterials. In this century, although
there are a number of recent papers concerning new monomers15 for this polymerization,
there are only a few reports concerning new initiators.16
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Recently, we unexpectedly found that aliphatic poly(ester-sulfone) prepared via
thiol-ene click polymerization and subsequent oxidation showed anode-selective
electrophoresis under EPD conditions, even though poly(ester-sulfone) does not have
any charge.17a Moreover, anode-selective EPD coating of bioactive glass,17a titanium
dioxide (TiO2)17b were also demonstrated. These interesting observations and behavior
prompted us to explore why the poly(ester-sulfone) is deposited on the anode, and what
part of the structure is responsible for the electrophoretic behavior.
We recently synthesized sulfide-containing methacrylates by esterification of
methacryloyl chloride with 2-(ethylthio)ethanol.18 Copper (I)-catalyzed atom transfer
radical polymerization (ATRP) of the sulfide-containing methacrylate was then carried
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out, and subsequent oxidation yielded electrophoritic vinyl polymers containing pendent
sulfone,18 which were compared to poly(ester-sulfone). In the former, the ester and
sulfone functionalities are both part of the main chain, wheares for the latter they are
part of the side chain. The sulfone-containing polymethacrylate also showed
anode-selective EPD behavior,18 which prompted an exploration of other types of
electrophoretic polymers containing sulfones.19
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This paper describes the preparation of a new initiator, N-methyl
bis[(nonafluorobutane)sulfonyl]imide (Nf2NMe), for the ring-opening polymerization
(ROP) of 2-oxazolines (Scheme 1), and compares it to a conventional initiator, methyl
triflate (TfOMe). In our laboratory, bis[(nonafluorobutane)sulfonyl]imide (Nf2NH) and a
silyl derivative were used as a catalyst for the polycondensation of diols and dicarboxylic
acid,20a-c ring-opening polymerization of ε-caprolactone,20d and aldol-type of
group-transfer polymerization,20e because of their high activity property. Furthermore,
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synthesis of electrophoritic poly(2-oxazoline)s containing pendent sulfone via ROP of
the vinyl-containing 2-oxazoline [2-(3-butenyl)-2-oxazoline] followed by a thiol-ene
click reaction and oxidation is demonstrated. Anode-selective electrophoretic coating of
the sulfone-containing poly(2-oxazoline) was investigated, and biomineralization
(appearance of hydroxyapatite) was confirmed on a poly(sulfonyl-oxazoline) / bioactive
glass composite via scanning electron microscopy (SEM). In addition, the cell
compatibility of poly(2-oxazoline)s containing pendent sulfone was evaluated using
human umbilical vein endothelial cells (HUVECs).
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EXPERIMENTAL SECTION
Materials and Methods.
Bis(nonafluorobutanesulfonyl)imide (Nf2NH) was
purchased from Mitsubishi Materials Electronic Chemicals (Akita, Japan).
2-ethyl-2-oxazoline, 2-phenyl-2-oxazoline, methyltriflate and 2-chloroethylamine
hydrochloride were purchased from Aldrich Chemical Co. (St. Louis, MO, USA).
Dimethyl sulfate was purchased from Tokyo Chemical Industry (Tokyo, Japan).
Dichloromethane (CH2Cl2), methanol (MeOH), diethyl ether (Et2O) and tetrahydrofuran
(THF) were purchased from Kanto Chemical (Tokyo, Japan). 4-Pentenoyl chloride,
1-propanethiol, CHCl3, N, N-dimethylformamide, potassium hydroxide (KOH) and
acetonitrile (CH3CN) were purchased from Wako Pure Chemical Industries, Ltd (Osaka,
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Japan). Methyl triflate, 2-oxazolines and all solvents were distilled prior to use. H
NMR spectra were acquired at 27˚C using a Bruker DPX200 spectrometer (200 MHz).
Tetramethylsilane was used as an internal standard (0 ppm). The FT-IR spectrum of each
polymer in KBr disks was obtained using a JASCO FT/IR-430 spectrometer. The number
average molecular weight (Mn) and the polydispersity index (Mw/Mn) of each polymer
were determined by size exclusion chromatography (SEC) using a Tosoh DP8020 pump
system, a refractive index (RI) detector (Tosoh RI-8020), and either a TSKgel
SuperMultiporeHZ-M column (eluent, chloroform; flow rate, 0.35 mL/min; temperature,
40 °C; Tosoh Corp.) or a TSKgel -3000 column [eluent, 0.05% (w/v) LiBr/DMF; flow
rate, 0.5 mL/min; temperature, 40 °C; Tosoh Corp.]. The columns were calibrated with
polystyrene standards.
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Preparation of Nf2NMe. Nf2NH (5.00 g, 8.60 mmol), and dimethyl sulfate (16.27 g,
0.13 mol) were added to a 50-mL round-bottom flask. The mixture was stirred at 100˚C
for 7 h under nitrogen. The mixture was then stirred at room temperature for 1 h, filtered,
and washed with CH3CN. After evaporation of the solvent, Nf2NMe was obtained as a
white solid in 60% yield (3.07 g). 1H NMR (200 MHz, CDCl3, δ): 3.63 (s, 1H) (see also
Figure S1), 19F NMR (400 MHz, CDCl3, δ): -81.66 (m, 3F), -107.63(m, 2F), -121.71(m,
2F), -126.74(brs, 2F) (see also Figure S2).21
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Preparation of 2-(3-Butenyl)-2-oxazoline. 2-(3-Butenyl)-2-oxazoline was
synthesized via N-(2-Chloroethyl)-4-pentenamide according to reported method22, 23 as
follows.
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N-(2-chloroethyl)-4-pentenamide: 4-Pentenoyl chloride (0.091 mol, 10.74 g) was
dissolved in 150 mL of dry CH2Cl2. 2-Chloroethylamine hydrochloride (0.091 mol, 10.5
g) was added, and the mixture was ice-cooled. Over a period of 1h, 40 mL of TEA was
added dropwise. After additional stirring for 30 min at 0 oC, the mixture was allowed to
room temperature and stirred overnight. After being stirred overnight, the organic layer
was separated and washed twice with 30 mL of distilled water and once with 30 mL of
brine. The combined fractions were dried over magnesium sulfate (MgSO4) and solvent
evaporated to give N-(2-chloroethyl)-4-pentenamide as a yellowish oil in 88% yield
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(9.28 g). H NMR (200 MHz, CDCl3, δ): 2.23-2.47 (4H, CH2=CH-CH2CH2-), 3.61 (4H,
-NH-CH2CH2-Cl), 4.92-5.19 (2H, CH2=CH-), 5.68-5.98 (1H, CH2=CH-), 6.11 (1H,
-NH-CH2-).
2-(3-Butenyl)-2-oxazoline: In a flame-dried flask, N-(2-chloroethyl)-4-pentenamide
(0.058 mol, 9.28g) was dissolved in 40 mL of dry MeOH. To this solution, a solution of
KOH (0.075 mol, 1.3 equiv) in 40 mL of dry MeOH was added drop by drop. The
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raction mixture was stirred for 12 h at 70 C under a nitogen atmosphere. The salt was
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filtered off and the remaining solution was concentrated in vacuo. Additionally
precipitated salt was removed by passing the solution through a 0.45 μm filter. The
product was distilled to give 2-(3-butenyl)-2-oxazoline as a colorless liquid in 34% yield
(2.42 g). 1H NMR (200 MHz, CDCl3, δ): 2.37 (4H, CH2=CH-CH2CH2-), 3.82 (t, 2H,
J=9.3 Hz, =N-CH2CH2-), 4.22 (t, 2H, J=9.3 Hz, -O-CH2CH2-), 4.95-5.14 (2H,
CH2=CH-), 5.73-5.97 (1H, CH2=CH-).
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Cationic Ring-opening Polymerization of 2-Oxazolines Initiated by
Nf2NMe.
Nf2NMe (0.0792 mmol, 47.1 mg) was stirred in a dry, 10-mL round-bottom
flask with CH3CN and either 2-ethyl-2-oxazoline (EtOx), 2-phenyl-2-oxazoline (PhOx),
or ButenylOx [3.96 mmol each; initial monomer concentration ([M]0): 1 M;
[M]0/[I]0=50] at various temperatures for various lengths of time under nitrogen (Table 1).
The reaction mixture was then allowed to cool to room temperature, and piperidine
(0.24 mmol, 3 equiv) was added to terminate the reaction. The mixture was stirred at
room temperature overnight, dry potassium carbonate (0.396 mmol, 5 equiv) was added,
and the mixture was again stirred overnight. After filtration, the polymer was purified
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by precipitation into excess amount of Et2O. H NMR (200 MHz, CDCl3, δ):
poly(2-ethyl-2-oxazoline) : 0.99-1.22 (br, -CH2CH3), 2.19-2.50 (br, -CH2CH3),
3.22-3.59 (br, -NCH2CH2N-). poly(2-phenyl-2-oxazoline)
:
2.55-3.90 (br,
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-NCH2CH2N- ), 6.82-7.17, 7.17-7.54 (br, -C=OC6H5). poly[2-(3-butenyl)-2-oxazoline] :
2.15-2.56 (br, -C=OCH2CH2-), 3.19-3.64 (br, -NCH2CH2N-), 4.89-5.16 (br, -CH=CH2),
5.65-5.98 (br, -CH=CH2). poly[(2-(3-butenyl)-2-oxazoline)-co-(2-ethyl-2-oxazoline)] :
0.98-1.23 (br, -CH2CH3), 2.17-2.56 (br, -CH2CH3 of poly(2-ethyl-2-oxazoline),
-C=OCH2CH2- of poly[2-(3-butenyl)-2-oxazoline], 3.22-3.67 (br, -NCH2CH2N- of
poly[(2-(3-butenyl)-2-oxazoline) and poly(2-ethyl-2-oxazoline) ) 4.91-5.15 (br,
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-CH=CH2),
5.67-5.97
(br,
-CH=CH2).
2.21-2.59 (br,
poly[(2-(3-butenyl)-2-oxazoline)-co-(2-phenyl-2-oxazoline)]
:
-C=OCH2CH2- of poly[(2-(3-butenyl)-2-oxazoline)), 2.71-3.85 (br, -NCH2CH2N- of
poly[(2-(3-butenyl)-2-oxazoline) and poly(2-phenyl-2-oxazoline)] 4.90-5.18 (br,
-CH=CH2), 5.68-5.99 (br, -CH=CH2), 6.92-7.19, 7.19-7.52 (br, -C=OC6H5).
Kinetic
plots
of
cationic
ring-opening
polymerization
of
2-ethyl-2-oxazoline. Two different Initiators [Nf2NMe (0.0792 mmol, 47.1 mg) or
TfOMe (0.0792 mmol, 8.96 μL) were stirred in each dry, 10-mL round-bottom flask with
CH3CN and either 2-ethyl-2-oxazoline (EtOx) [3.96 mmol each; initial monomer
o
concentration ([M]0): 1 M; [M]0/[I]0=50/1] at 80 C for various lengths of time under
nitrogen. During the polymerization, samples were taken from the reaction mixtures to
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sample vials prefilled with chloroform at suitable time intervals. Subsequently,
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piperidine was added to terminate the reactions. kp were calculated by using eq (1)
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[
]
[ ]
(1)
[ ] ･
Thiol-ene
Click
Reaction
of
Poly[2-(3-butenyl)-2-oxazoline].
Poly[2-(3-butenyl)-2-oxazoline] (330 mg, 2.63 mmol 1 equiv) was dissolved in 9.1 mL of
dry THF. 1-Propanethiol (0.29 mL, 3.2 mmol, 1.2 equiv) and 1-hydroxycyclohexyl
phenyl ketone (Irgacure 184) (25.7 mg, 0.13 mmol) were added, and the mixture was
degassed and replaced with nitrogen three times, and exposed to UV light using a
Toshiba H-400P high-pressure mercury lamp (400 W and 250-400 nm) for 2 h (Scheme
2). After evaporation of the solvent and excess 1-propanethiol, the sulfide-containing
1
poly(2-oxazoline) was obtained (yield: 503 mg, 95%). H NMR (200 MHz, CDCl3, δ) :
0.98 (t, -CH2CH3, 7.3 Hz), 1.45-1.81 (br, -CH2CH3, -C=OCH2CH2, -C=OCH2CH2CH2),
2.18-2.42 (br, -C=OCH2-), 2.42-2.61 (br, -CH2SCH2-), 3.31-3.56 (br, -NCH2CH2N-).
Oxone Oxidation24 of Poly(2-oxazoline)s Having Pendant Sulfide to
Sulfonyl Poly(2-oxazoline). Sulfide-containing poly(2-oxazoline) (201 mg, 1.00
mmol) was dissolved in 5 mL of CHCl3. Oxone® (922 mg, 1.5 mmol, 1.5 equiv)24 was
dissolved in 20 mL of distilled water. The two solutions were then combined and stirred
at room temperature for 48 h. After 48 h, the CHCl3 layer was washed twice with
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distilled water, and the organic phase was evaporated to give the expected sulfonyl
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poly(2-oxazoline) (yield: 239 mg, 90 %). H NMR (200 MHz, CDCl3, δ): 1.07 (t,
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-CH2CH3, 7.4 Hz), 1.62-1.97 (br, -CH2CH3, -C=OCH2CH2, -C=OCH2CH2CH2),
2.27-2.57 (br, -C=OCH2-), 2.85-3.13 (br, -CH2SO2CH2-), 3.25-3.69 (br, -NCH2CH2N-).
Electrophoretic Deposition of Sulfonyl Poly(2-oxazoline)． The depositions
were carried out on 0.2 × 15 × 25 mm stainless steel SUS 301 electrodes obtained from
The Japan Metal Service Corporation (Saitama, Japan). To deposit the sulfonyl
poly(2-oxazoline) composite onto the stainless steel electrode via EPD, the distance
between the electrodes was 6.5 mm, and the deposition time was 90 s at a voltage of 250
V.
Electrophoretic Deposition of Sulfonyl Poly(2-oxazoline)/Bioglass®45S5
Composites. Bioglass®45S5 powder (45 wt% SiO2, 24.5 wt% Na2O, 24.5wt% CaO,
and 6 wt% P2O5) of mean particle size < 7 μm was used. A suspension of the
Bioglass®45S5 powder in n-butanol was agitated in an ultrasonicator for 15 min and then
stirred for 5 min. The sulfonyl poly(2-oxazoline) was stirred in DMF for 15 min to obtain
a homogeneous solution. The Bioglass®45S5 suspension was then dispersed in the
sulfonyl poly(2-oxazoline) solution, and the mixture was stirred for 5 min. The
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Bioglass®45S5/sulfonyl poly(2-oxazoline) composite was then deposited onto the same
type of stainless steel electrodes via EPD, as above.
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Biomineralization of Bioactive Glass / Sulfonyl Poly(2-oxazoline) Composite Film.
The surface composition of the Bioglass®45S5/sulfonyl poly(2-oxazoline) composite
coating deposited on the anode was investigated after immersion of the samples in 30 mL
of simulated body fluid (SBF) buffered with tris[hydroxymethyl]aminomethane and
hydrochloric acid to maintain a pH of 7.4 at 37C. The composition of SBF has been
described in the literature.25 After 2 weeks, the composites were taken out and washed
with distilled water. The surface of the Bioglass®45S5/sulfonyl poly(2-oxazoline)
composite film was visualized via scanning electron microscopy (SEM) (dual-stage
JSM-6010LA; JEOL Ltd., Tokyo, Japan). Elemental composition was also investigated
via energy-dispersive X-ray spectroscopy (EDX). The elemental distribution was
determined using Smart Map, which simultaneously acquires X-ray data from each pixel
in an image (JEOL Ltd., Tokyo, Japan). X-ray diffraction (XRD) was used to determine
the crystalline composition of the film surfaces and was performed with an automated
diffractometer (RINT2000/PC; Rigaku, Tokyo, JAPAN) at a scan rate of 2 min–1,
between 10 and 55.
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Cell Compatibility of Sulfonyl Poly(2-oxazoline). Sulfonyl poly(2-oxazoline) (27
mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (DMF) (1.00 mL). The
solution was dropped onto a cover glass (22 mm × 22 mm). The cover glass was
spin-coated at 1000 rpm for 300s on a MIKASA 1H-D7 spin coater and dried. The
spin-coating was repeated 5 times. Procedures of cell culture and spreading are as
follows.
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Cell Culture: HUVECs (Lonza Walkersville, Inc.) were maintained in a 37 C, 5% CO2
humidified environmental chamber. The cells were grown in Endothelial Growth
Medium-2 (EGM-2, 2% FBS; Lonza Walkersville), which was replaced every 2 days.
Near confluent HUVEC cultures were passaged by treatment with 0.025%
Trypsin/0.01% EDTA (Lonza Walkersville).
Cell Spreading: HUVECs in EGM-2 were seeded on sulfonyl poly(2-oxazoline) coated
cover glass in 40×15 mm Petri dish at a concentration of 5000 cells per Petri dish. Cells
were imaged using a 10×phase contrast objective on a Nikon Eclipse TS100/ TS100-F
epifluorescence microscope (Tokyo, Japan).
RESULTS AND DISCUSSION
ROP of 2-Oxazolines. 2-Oxazoline containing pendent vinyl groups was
prepared via N-(2-chloroethyl)-4-pentenamide using 4-pentenoyl chloride and
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2-chloroethylamine hydrochloride as the starting materials (34% yield) using the
literature procedure.22, 23 This study evaluates the use of the methylated derivative of
Nf2NH, N-methyl bis[(nonafluorobutane)sulfonyl]imide (Nf2NMe) as a new initiator for
the cationic ring-opening polymerization of 2-oxazolines. The results are summarized in
Table 1. The structure of the polymer was confirmed by 1H NMR (Figure S3) and SEC
(Figure S4). The polymerization activity and the control of molecular weight of Nf2NMe
as the new initiator for polymerization of 2-ethyl-2-oxazolines (EtOx) were investigated
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first. The cationic ring-opening polymerization of EtOx in acetonitrile at 80 C with
Nf2NMe as the initiator was compared with methyl triflate (TfOMe), and the two had
similar polymerization activity (Figure 1). Also, we calculated both kp using eq (1) in
experimental section (Nf2NMe : kp = 14.6 ± 0.40×10-4 L/mol･s, TfOMe : kp = 14.1 ±
0.42 × 10-4 L/mol･s). In addition, the livingness of the propagating site was evaluated in
a two-step polymerization, and the propagating center was identified as the oxazolinium
ion by 1H NMR spectroscopy (Figure S3).
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Nf2NMe
TfOMe
0
2
4
6
Time (h)
Figure 1. ln[M]0/[M] vs time plots of polymerization of 2-ethyl-2-oxazoline (1 M)
initiated by Nf2NMe and TfOMe ([M]0/[I] = 50/1, in CH3CN, at 80 oC).
Next, the homopolymerizations of PhOx and ButenylOx were also undertaken,
as well as the ring-opening block copolymerization of ButenylOx/EtOx and
ButenylOx/PhOx (Table 1), all with Nf2NMe as the new initiator as shown in Scheme 1.
The polymerization was performed using a one-pot, two-stage method.19 The ROPs
proceeded as expected, and the structures of the homopolymers (Figure S6 and S7) and
1
block copolymers were confirmed by H NMR (Figure 2 and Figures S8). The
polymerization results are also summarized in Table 1.
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Table 1. Ring-Opening Polymerization of 2-Oxazolines Initiated by Nf2NMea
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1st stage
2nd stage
Conv. b
Yield d
Monomer
second eq.
M n×10-3 c M w/M n
c
Unit ratio b
Unit ratio b
Run
Time
Time
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first
EtOx
PhOx
(h)
36
96
36
(x : y)
50 : 0
50 : 0
50 : 0
25 : 0
25 : 0
(h)
-
-
(x : y)
50 : 0
50 : 0
50 : 0
23 : 27
22 : 28
(%)
>99
88
>99
>99
93
(%)
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74
93
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50+0
50+0
50+0
2.7
4.1
3.3
3.6
4.7
1.29
1.24
1.26
1.30
1.29
ButenylOx
-
ButenylOx EtOx 25+25 24
ButenylOx PhOx 25+25 24
16
113
88
[M]0/[I]0=50/1. Determined by H NMR spectroscopy; solvent, CDCl3. c Determined by
SEC measurement in CHCl3 relative to poly(styrene)s. d After purification by reprecipitation.
b
1
a
Figure 2. 1H NMR spectrum of poly[(2-(3-butenyl)-2-oxazoline-block-
(2-ethyl-2-oxazoline)] (in CDCl3, 27 C).
Oxone Oxidation of the Poly(2-oxazoline) Having Pendent Sulfide to the
Sulfonyl Poly(2-oxazoline). A thiol-ene click reaction of the poly(2-oxazoline) having
a pendent vinyl group with 1.2 eq of propanethiol was accomplished in THF to give
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expected parent sulfide poly(2-oxazoline) (Figure S9). Subsequently, oxone (1.5 eq. to
sulfide) oxidation of the poly(2-oxazoline) having a pendent sulfide group was carried
out in CHCl3 / H2O at room temperature for 48 h (Scheme 2). The expected structure was
confirmed via 1H NMR and IR (Figures 3 and 4, respectively). The oxidation conversions
(>99%) were estimated using the intensity ratio of the signals at 2.42-2.61 ppm (br,
-CH2SCH2-) and 2.85-3.13 ppm (br, -CH2SO2CH2-) in the 1H NMR (Figure 3).
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Figure 3. 1H NMR spectrum of sulfonyl poly(2-oxazoline) (in CDCl3, 27 C).
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Figure 4. FT-IR spectra of (a): sulfide poly(2-oxazoline) and (b): sulfonyl
poly(2-oxazoline). Sulfonyl groups were confirmed by the presence of signals at 1123
and 1281 cm-1.
Preparation of Stainless Steel Electrodes Coated with Electrophoretic
Poly(2-oxazoline) and Bioglass®45S5/ Sulfonyl Poly(2-oxazoline) Composites.
Sulfonyl poly(2-oxazoline) (Mn = 8.6×103) alone, as well as mixtures with
Bioglass®45S5 form colloidal suspensions in mixed solvents of DMF and n-butanol,
and they are both deposited onto a stainless-steel anode by EPD (Table 2).
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Table 2. Bioglass®45S5/Sulfonyl Poly(2-oxazoline) Coating on the Stainless Steel
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Electrode by EPD a
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Polymer/Bioglass®45S5
(mg/mL)
Image of the
Solvent
M n×10-3 b
Run
Content
Electrode
electrode deposit
DMF/n-BuOH (mL/mL)
15
Bioglass®45S5
1
0
6
-
2 / 2
cathode
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-
Sulfide poly(2-oxazoline)
Sulfonyl poly(2-oxazoline)
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0.5 / 2.75
anode
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8.6
anode
Sulfonyl poly(2-oxazoline)
/Bioglass®45S5
a
b
Deposition time of 90 seconds, at a voltage of 250 V. Determined by GPC
measurement in DMF relative to poly(styrene)s.
Only Bioglass®45S5 particles moved toward the cathode in this system (run 1)
because the surface of these particles is covered with Ca2+ and Na+.4c The parent sulfide
poly(2-oxazoline) (Figure S9) was not deposited on either substrate (run 2). Conversely,
deposition of pure sulfonyl poly(2-oxazoline) was achieved via electrophoretic motion of
the polar sulfone in suspension to the anode (“electrophoretic polyoxazoline,” run 3). It is
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likely that polarized sulfones dominate the surface charge of the composite (Figure 5).
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The H NMR spectra (Figure 6) of the sulfonyl poly(2-oxazoline) before and after
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deposition on the electrode (run 3) indicate that EPD did not alter the
structure/composition of the sulfonyl poly(2-oxazoline). A composite of Bioglass®45S5
and sulfonyl poly(2-oxazoline) was fabricated via EPD, and this Bioglass®45S5/sulfonyl
poly(2-oxazoline) composite was also deposited on the anode substrate (run 4). Therefore,
control of the electrophoretic behavior of a sulfonyl poly(2-oxazoline) can probably be
accomplished by hybridization with Bioglass®45S5 via EPD.
Figure 5. (a) Schematic of a charged Bioglass®45S5/sulfonyl poly(2-oxazoline)
colloidal
electrophoretically-controlled
suspension
in
organic
deposition
solution.
of
(b)
the
Schematic
of
the
Bioglass®45S5/Sulfonyl
poly(2-oxazoline) colloid. (Adapted with permission from ref 17a. Copyright 2012
American Chemical Society.)
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0
Chemical Shift (ppm)
1
Figure 6. H NMR spectra of sulfonyl poly(2-oxazoline) before (black) and after
(red) EPD.
Biomineralization of Bioglass®45S5/Sulfonyl Poly(2-oxazoline) Having Pendent
Sulfones Composite Films. The surface composition of the Bioglass®45S5/sulfonyl
poly(2-oxazoline) composite deposited on the anode was investigated after immersion of
the samples in SBF. The surface of the Bioglass®45S5/sulfonyl poly(2-oxazoline) film
before and after immersion in SBF was evaluated via SEM (Figure 7). Images of the film
immersed in SBF reveal a dense deposit covering the film [Figure 7(b)] which was absent
prior to soaking [Figure 7(a)]. The morphology of these deposits is very similar to that of
hydroxyapatite (the chief component of bones and teeth) deposited on titanium or a
bioactive glass/ceramic composite by biomimetic methods, i.e. upon exposure to SBF.4a,
4i, 25 The surface morphology was further investigated via EDX (Figure 8). The colors in
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the EDX map identify carbon (red), calcium (green), and phosphorus (blue), and show
that the surface is almost entirely covered by calcium and phosphorus, components of
hydroxyapatite. The diffraction pattern of the XRD profile (Figure 9) is also indicative
of hydroxyapatite.17a Thus, it is clear that hydroxyapatite was formed on the
Bioglass®45S5/sulfonyl poly(2-oxazoline) composite film after immersion in SBF for 2
weeks.
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Figure 7. Scanning electron micrographs of Bioglass®45S5/sulfonyl
poly(2-oxazoline) films. (a): before immersion in SBF (magnification 500) (b): after 2
weeks of immersion at 37 C (magnification 7500).
Figure 8. EDX map showing the distribution of carbon (red), calcium (green), and
phosphorus (blue) on Bioglass®45S5/sulfonyl poly(2-oxazoline) film after immersion in SBF
for 2 weeks (magnification ×7500).
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Figure 9. XRD patterns for the Bioglass®45S5/sulfonyl poly(2-oxazoline) film (a):
before immersion in SBF. (b): after immersion in SBF for 2 weeks.
Cell Compatibility of Sulfonyl Poly(2-oxazoline).
Finally, the cell
compatibility of sulfonyl poly(2-oxazoline) was evaluated to assay its utility as a new
biomaterial. In previous reports, poly(2-oxazoline)s such as poly(2-methyl-2-oxazoline)
and poly(2-ethyl-2-oxazoline) were shown to be biocompatibile,13a,26 and we expected
the same for sulfonyl poly(2-oxazoline). HUVECs plated on sulfonyl polyoxazoline
films were monitored at 1 day intervals by phase contrast microscopy and were
categorized as dark (spread) (Figure 10). The assay was accomplished by investigating
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the viability of cell (HUVEC) proliferation on a spin-coated sulfonyl poly(2-oxazoline)
film on a glass plate. The cells adhered to the sulfonyl poly(2-oxazoline)-coated glass
plates within a day. Even after 4 days at biological temperature (37 C), the surface did
not show detectable toxicity toward the HUVECs (Figure 10c). The number of
proliferized cells (dark part in Figure 10c) on the oxazoline coating increased 10 times
within 4 days after cell seeding (Figure 11). These results were comparable to a positive
control (cultivation) using a commercially-available cell cultivation dish (Figure 10b,
Figure 11). On the blank (negative control) using normal polystyrene dish, increase of
cell number or any proliferations (dark part) were not observed. From the results, we
concluded sulfonyl poly(2-oxazoline) has biocompatibility with HUVECs.
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Figure 10. Cell morphology (a) blank, (b) positive control [cell cultivation dish], (c)
sulfonyl poly(2-oxazoline).
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[×103]
40
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positive control
(cell cultivation
dish)
sulfonyl poly(2-
oxazoline)
0
0
1
2
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4
Time (d)
Figure 11. Proliferation of HUVECs on sulfonyl poly(2-oxazoline) coating.
CONCLUSION
In this study, a new initiator, Nf2NMe, was synthesized for the polymerization
of 2-oxazolines. It has similar polymerization activity to that of the conventional initiator,
and demonstrates controlled polymerization. Poly(2-oxazoline)s having pendent sulfones
were prepared and deposited onto a stainless steel surface via EPD, and sulfonyl
poly(2-oxazoline) shows anode selective behavior. Moreover, EPD was successfully
used to hybridize Bioglass®45S5 and sulfonyl poly(2-oxazoline). Hydroxyapatite was
confirmed to form on the surface of a Bioglass®45S5/sulfonyl poly(2-oxazoline)
composite film immersed in SBF for 2 weeks at biological temperature. This new
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sulfonyl poly(2-oxazoline) has a biocompatibility similar to poly(2-methyl-2-oxazoline)
and poly(2-ethyl-2-oxazoline). Because of this, electrophoretic sulfonyl
poly(2-oxazoline) could find application in biomaterials such as implants, bone
regeneration materials and cell scaffolds. The results support our recent finding that
poly(ester-sulfone)s prepared via click polymerization and a thiol-ene click reaction
show anode-selective deposition behavior, and they also provide a new pathway for the
production of a biomaterial utilizing the characteristics of poly(2-oxazoline)s.
Supporting information available
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Detailed experimental section, 1H NMR, 19F NMR spectra of Nf2NMe, 1H NMR spectra
of produced poly(2-oxazoline)s and molecular weight (Mn), Mw/Mn against conversion
plot of polymerization of 2-ethyl-2-oxazoline initiated by Nf2NMe. This information is
available free of charge via the Internet at http://pubs.acs.org/.
AUTHOR INFORMATION
Corresponding Author: Akinori Takasu
*Tel: +81-52-735-7159. Fax: +81-52-735-5342. E-mail: takasu.akinori@nitech.ac.jp
ACKNOWLEDGMENTS
This work was funded by the Ministry of Education, Science, and Culture of Japan
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(Grant-in-Aid for Development Scientific Research, no. 24550132). The authors are
grateful to Professor Aldo R. Boccaccin, Institute of Biomaterials, University of
Erlangen-Nurnberg, for providing Bioglass®45S5 and fruitful discussions.
REFERENCES
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