synthesis and characterization of amphiphilic copolymers with...
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공학 사학 논문
양쪽 온 및 식물 래 카다놀 포함한 양친 고분 합
및 특 분 , 그리고 방오 및 항균 코 재로 용
Synthesis and Characterization of Amphiphilic
Copolymers with Zwitterionic and Cardanol Moieties for
Antifouling and Antibacterial Coating Applications
2016년 2월
울 학 학원
화학생물공학부
다
i
Abstract
Synthesis and Characterization of Amphiphilic Copolymers with
Zwitterionic and Cardanol Moieties for Antifouling and
Antibacterial Coating Applications
Dajung Shon
Polymer Chemistry
Department of Chemical & Biological Engineering
Seoul National University
A series of copolymers [PSH#s, where # means a mol % of sulfobetaine
methacrylate (SBMA) unit in the polymers] with zwitterion and cardanol moieties
were synthesized via free radical polymerization of 2-(dimethylamino)ethyl
methacrylate (DMAEMA) and plant-derived monomer, 2-hydroxy-3-
carbonylpropyl methacrylate (HCPM), followed by betainisation with 1,3-propane
sultone.
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Cross-linked PSH# (PSH#C) films were prepared through spin-coating or drop-
casting method and then UV light irradiation, and their surface properties were
investigated. The PSH#C films showed high transparency to the visible light
region, and a water static contact angle of their surfaces decreased when the
amount of hydrophilic SBMA unit in the copolymers increased.
Antifouling characteristics against both Escherichia coli (E.coli) and bovine
serum albumin (BSA) of the PSH#C films were enhanced as the SBMA moiety in
the copolymers increased. All of the PSH#C films were non-cytotoxic against
Human dermal fibroblasts (HDFs) and the amount of HDFs attached to the
surfaces decreased when the zwitterion moiety in the PSH increased. Furthermore,
the PSH#C films exhibited high antibacterial property against E.coli because of a
presence of the bactericidal HCPM moiety in the PSH.
Consequently, it was demonstrated that the PSHs have good anti-adhesion and
bactericidal characteristics due to the SBMA and HCPM respectively, and non-
cytotoxicity. Especially, PSH42 having proper amount of the SBMA and HCPM
units exhibited excellent antifouling and antibacterial properties, and is expected
iii
to be a candidate for coating material in anti-biofouling fields.
Keywords: Zwitterion, cardanol, antifouling, bactericidal, coating material
Student number: 2014-20564
iv
List of Tables
Table 1. Synthesis results of the PDH#s from different co-monomers feeding
ratios.
Table 2. Elemental analysis of the PSHs.
Table 3. XPS analysis of the PSH#C films.
v
List of Scheme and Figures
Scheme 1. Synthesis of (a) 2-hydroxy-3-cardanolpropyl methacrylate (HCPM), (b)
poly(DMAEMA-co-HCPM) (PDM), and (c) poly(SBMA-co-HCPM)
(PSH).
Fig. 1 1H NMR spectra of (a) HCPM, (b) PDMAEMA, (c) PHCPM(PSH0) and (d)
PSH24.
Fig. 2 TGA thermograms of the PSHs under N2 atmosphere.
Fig. 3 DSC thermograms of the PSHs under (a) 1st heating condition and (b) 1st
heating condition (PSH0, PSH21 and PSH42) and 2nd heating condition
(PSH68 and PSH94).
Fig. 4 FT-IR/ATR spectra of PSH0C film in the high frequency region after UV
irradiation for 2 hours.
Fig. 5 UV-Vis spectra of cross-linked PSH (PSH#C) films on glass substrates.
Inset is photograph of the PSH#C films prepared by a spin coating method.
Fig. 6 Water static contact angles of the PSH#C films. Inset is photographic
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results.
Fig. 7 Representative fluorescence microscope images of adhered E.coli onto the
PMMA and PSH#C films.
Fig. 8 Representative fluorescence microscope images of adhered BSA onto the
PMMA and PSH#C films (scale bar = 100 µm).
Fig. 9 Representative optical microscope images of HDFs cultured for (a) 2 and (b)
4 days on the surfaces of the PMMA and PSH#C films (scale bar = 100 µm).
Cell viability determined by MTT assay after (c) 2 and (d) 4 days.
Fig. 10 CLSM images presenting FDA/EB staining on HDFs cultured for (a) 2
and (b) 4 days on the PMMA and PSH#C films (scale bar = 100 µm).
Fig. 11 (a) Photographs of the PMMA and PSH#C films via antibacterial test
against E.coli. (b) Bactericidal activity of the PMMA and PSH#C films.
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List of Contents
Abstract ------------------------------------------------------------------------------------ⅰ
List of Tables ----------------------------------------------------------------------------- ⅳ
List of Scheme and Figures ----------------------------------------------------------- ⅴ
List of Contents ------------------------------------------------------------------------- ⅶ
1. Introduction ------------------------------------------------------------------------- 1
2. Experimental ------------------------------------------------------------------------ 4
2.1. Materials ------------------------------------------------------------------------- 4
2.2. Synthesis of 2-hydroxy-3-cardanylpropyl methacrylate (HCPM) --- 6
2.3. Synthesis of copolymers containing HCPM and DMAEMA
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monomeric units (P(DMAEMA-r-HCPM), PDHs) -------------------- 7
2.4. Betainisation reaction of PDHs ---------------------------------------------- 9
2.5. Preparation of cross-linked PSH (PSH#C) films ----------------------- 10
2.6. Antifouling test ---------------------------------------------------------------- 11
2.7 Anti-HDF fouling and cytotoxicity test ------------------------------------ 12
2.8 Antibacterial test --------------------------------------------------------------- 14
2.9. Characterization -------------------------------------------------------------- 16
3. Results and Discussion ---------------------------------------------------------- 19
3.1. Synthesis of 2-hydroxy-3-cardanylpropyl methacrylate (HCPM) --- 19
3.2. Synthesis of copolymers with DMAEMA and HCPM moieties ------ 19
3.3. Betainisation reaction of the PDHs ---------------------------------------- 25
3.4. Preparation of cross-linked PSH# (PSH#C) films ---------------------- 30
3.5. Optical and chemical characteristics of the PSH#C films ------------- 33
3.6. Antifouling properties of the PSH#C films ------------------------------- 36
ix
3.7. Antifouling and cytotoxic characteristics of the PSH#C films against
HDFs ---------------------------------------------------------------------------- 39
3.8. Antibacterial properties of the PSH#C films ---------------------------- 46
4. Conclusion --------------------------------------------------------------------------- 49
5. References ---------------------------------------------------------------------------- 51
6. Abstract in Korean ----------------------------------------------------------------- 59
1
1. Introduction
There are many microbial fouling phenomena around human being life.
Biofouling, undesirable accumulation of micro- and macro- organisms by various
mechanisms1-5 can be a serious problem in shipping, medical, and water-treatment
industries. For example, the organisms attached to the vessel can increase a
hydrodynamic drag and it leads to the increase of a fuel consumption rate up to
40 %.6-8 Also, the biofouling cause microbiologically influenced corrosion (MIC)
due to life cycle of the marine organism and decomposition product generated by
them, and the MIC-related damages cost about 30-50 billion dollars annually as a
result.9-11 In the medical fields, the biofoulings attached on medical devices such
as catheters and implants can be detrimental to human and deteriorate function of
the devices.12-14 The microbial fouling on the membranes for water purification in
desalination system causes the decrease of the membrane lifetime, so an exchange
cycle of the membrane should be shorten.15-17 Therefore, there have been many
researches to prevent the formation of the problematic biofouling. There are two
2
approaches to inhibit the biofouling. One is to make an antifouling surface and the
other is to form a bactericidal surface.
Three major strategies have been researched to produce the antifouling surfaces.
Homogeneous surfaces are used to exhibit the anti-adhesion property using
hydrophobic, hydrophilic, and amphiphilic materials. Mixed or patterned surface
comprised by both hydrophilic and hydrophobic material, and 3D surfaces can
also be used for the antifouling surfaces.18 Among these methods, in an approach
of making the hydrophilic homogeneous surface, hydration layers formed by
interaction between water molecules and the hydrophilic surface play a role as a
barrier to inhibit the microorganism attachment. Poly(ethylene glycol) (PEG) has
been commonly used as the fouling resistant materials. Due to the chain flexibility
and the hydration layer formation by hydrogen bonding interaction with the water,
the PEG can prevent the fouled organisms effectively.8,12,17,19 However, the PEG
has drawbacks such as oxidative or thermal degradation, unexpected
pharmacokinetic changes, and non-biodegradability,20-25 so alternatives with
hydrophilic characteristic have been developed. Recently, zwitteroinic polymers
3
have been researched for the antifouling applications to replace the PEG due to
the excellent non-adhesion property without weakness of the PEG. The
zwitteroinic polymers have both positive and negative charges, but net charge is
zero because a ratio of these opposite charge is same. The zwitteroinic polymers
such as poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC),
poly(sulfobetaine methacrylate) (pSBMA), and poly(carboxybetaine methacrylate)
(pCBMA) develop the hydration layers through strong ionic interaction with the
water molecules instead of the hydrogen bonding, showing better antifouling
properties than the PEG as a result.26-39
There have been many researches about antibacterial materials such as
quarternary ammonium compound (QAC), silver nanoparticles, N-halamine and
so on.40-42 Among the bactericidal materials, the QAC is the most well-known
material with its excellent antimicrobial property, disrupting a cellular membrane
of the bacteria.8,43-47 Cardanol is also an example of the antibacterial material and
renewable material obtained from cashew nut shell liquid (CNSL) through
vacuum distillation.48-52 The cardanol have C15 unsaturated hydrocarbon chains
4
with one, two, and three double bonds at the meta position of the phenol group.
The cardanol has been used and studied for various applications such as coating,
varnishes, and brake linings. There have been also reports about the bactericidal
properties of the cardanol although the antibacterial mechanism of the cardanol is
unclear.53-55 Recently, systematic researches about the antimicrobial characteristics
of cardanol-based polymers were also studied.56
In this paper, copolymers with antifouling and bactericidal properties were
synthesized using zwitterion and cardanol and applied as a coating materials,
expecting the effects that surfaces coated with the polymer prohibit an attachment
of the foulants and kill the fouled micro-organisms. After surface coating using
the polymer with dual characteristics, various surface properties such as fouling
resistance and antimicrobial property were investigated.
2. Experimental
2.1. Materials
5
Cardanol was provided by Mercury Co., Ltd. (India). Glycidyl methacrylate
and1,3-propane sultone were purchased from TCI Co., Ltd. 2,2,2-trifluoroethanol
(TFE) was obtained from Alfa aesar Co., Ltd. Anisole (anhydrous, 99.7%),
phosphate buffered saline (PBS, powder, pH 7.4) and phosphonium iodide (PI)
were purchased from Sigma-Aldrich Co., Ltd. 2,2’-azobis(isobutyronitrile) (AIBN,
Junsei) was recrystallized from ethanol prior to use. 2-
(dimethylamino)ethylmethacrylate (DMAEMA, Sigma-Aldrich) was purified with
aluminum oxide (Al2O3, Acros organics Co., Ltd.) for removing inhibitors.
Tetrahydrofuran (THF, Daejung Chemicals & Metals) was dried by refluxing over
sodium and benzophenone, followed by distillation. Potassium hydroxide (KOH)
was obtained from Daejung Chemicals & Metals Co., Ltd. Escherichia coli
(E.coli, ATCC 8739) was obtained from American Type Culture Collection
(ATCC). BactoTM agar and DifcoTM nutrient broth were obtained from Becton,
Dickinson and Co. (BD). SYTO 9 was obtained from Life technologies Co., Ltd.
All other reagents were obtained from standard vendors and used as received.
6
2.2. Synthesis of 2-hydroxy-3-cardanylpropyl methacrylate (HCPM)
Cardanol (10 g, 33 mmol) and potassium hydroxide (KOH, 1.9 g, 33mmol) was
added to a 250 mL round-bottom flask containing magnetic stirring bar and
dimethylacetamide (DMAc, 30 mL) was poured under nitrogen (N2) atmosphere.
After glycidyl methacrylate (9.4g, 66 mmol) was dropped to the solution, the
mixture was stirred for 72 hours at 60 oC. The reaction was terminated when a few
drops of concentrated hydrogen chloride (HCl) solution were added to the reactor
and DMAc was evaporated in a low pressure and elevated temperature condition.
The crude product was dissolved in methylene chloride (MC) and transferred to a
separatory funnel for an extraction. After 0.5 N HCl solution was added to the
separatory funnel, MC layer was obtained. Moisture remained in the MC layer
was removed using anhydrous magnesium sulfate and the product was filtered.
The final product was purified through silica gel column chromatography (ethyl
acetate : n-hexane = 1 : 6 vol %). An orange-colored and viscous liquid was
7
obtained and a yield was 49 % (7.2 g). 1H NMR (300 MHz, CDCl3,
trimethylsilane (TMS) ref): δ = 0.88 (t, J = 6.78 Hz, 3H, – CH3), 1.20-1.40 (m,
CH3(CH2)12CH2–), 1.60 (m, 2H, CH3(CH2)12CH2CH2–), 1.97 (s, 3H, –
OC(O)C(CH3)=CH2), 2.02 (m, –CH2CH2CH2CH=CHCH2–), 2.57 (t, J = 8.04 Hz,
2H, – OC6H4CH2–), 2.75-2.90 (m, –CH2CH=CHCH2CH=CH–), 3.94-4.40 (m, 5H,
– OCH2CH(OH)CH2OC(O)–), 4.97-5.80 (m, –CH2CH=CHCH2–), 5.62 and 6.26
(s, 2H, – OC(O)C(CH3)=CH2), 6.67-6.83 (m, 3H, aromatic), 7.19 (t, J = 7.5 Hz,
1H, aromatic). FT-IR: 3471 cm-1 (O-H stretching vibration), 3010 cm-1 (C-H
vibration of the unsaturated hydrocarbon), 1720 cm-1 (C=O stretching vibration
(α,β-unsaturated ester), 1261 cm-1 (C(Ar)–O–C asymmetric stretching vibration
(m-alkyl phenol)), 1049 cm-1 (C(Ar)–O–C symmetric stretching vibration (m-
alkyl phenol)), 775 cm-1 (–CH2– rocking vibration), 721 cm-1 (–(CH2)n–, n > 3;
rocking vibration), 694 cm-1 (aromatic out of plane C–H deformation vibration of
m-substituted benzene). Mass m/z calculated C28H44O4+ : 444.32, found 444.0.
2.3. Synthesis of copolymers containing HCPM and DMAEMA
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monomeric units (P(DMAEMA-r-HCPM), PDHs)
The copolymers consisting HCPM and DMAEMA monomeric units were
abbreviated as PDH#, where # means the molar contents of the DMAEMA
monomer in the copolymers. The following procedure was an example synthesis
of PDH42 containing 42 mol% of the DMAEMA and 58 mol% of the HCPM
monomeric units. HCPM (1.5 g, 3.4 mmol), DMAEMA (0.35g, 2.3 mmol), AIBN
(0.019g, 0.11 mmol), and anisole (9.3 mL) were placed in a 50 mL round-bottom
flask containing magnetic stirring bar. The flask was degassed under N2
atmosphere for 20 minutes to remove dissolved oxygen and then the reaction was
continued at 70 oC with stirring. After 18 hours of the polymerization, the product
was exposed to the air to finish the reaction and poured into excess of
DIW/methanol (5/1) with stirring. The dissolution-precipitation process was
repeated thrice and yellowish wax product was obtained after drying under
vacuum condition (1.5g). Other PDHs were synthesized using same procedure
except the monomer feed ratio (DMAEMA : HCPM) (Table 1). 1H NMR (300
9
MHz, CDCl3, TMS ref): δ = 0.8–1.1 (3H, –CH3), 1.20–1.90 (m, CH3(CH2)12CH2–
and backbone), 1.55 (m, 2H,CH3(CH2)12CH2CH2–), 2.02 (m, –
CH2CH2CH2CH=CHCH2–), 2.25 (m, 6H, -NCH3), 2.51 (2H, –OC6H4CH2–),
2.75–2.90 (m, –CH2CH=CHCH2CH=CH–), 3.90–4.40 (m, 5H, –
OCH2CH(OH)CH2OC(O)–), 4.97–5.80 (m, –CH2CH=CHCH2–), 6.50–6.83 (m,
3H, aromatic), 7.13 (1H, aromatic).
2.4. Betainisation reaction of PDHs
Betainisation was carried out to functionalize the amine group of the
DMAEMA unit in the PDHs to zwitterionic group. The copolymers containing the
sulfobetaine methacrylate (SBMA) and HCPM moiety was designated as PSH#,
where # represents the molar ratio of the SBMA unit in the copolymers. A 10 mol%
excess 1,3-propane sultone of the DMAEMA portion was placed on a 50 mL
round-bottom flask with stirring bar inside argon (Ar) filled glove box and 2 wt%
PDH solution dissolved in THF was added to the flask. The product was obtained
10
after 24 hours at 25 oC and poured into an excess of DIW with stirring. The
dissolution-precipitation process was repeated three times and white powdery
product was obtained after drying under vacuum condition.
2.5. Preparation of cross-linked PSH (PSH#C) films
1 wt% of PSHs were coated on silicon wafer and glass substrates using spin-
coater and dried in a vacuum condition overnight. Chloroform (CHCl3) was used
for PSH0, PSH21, and PSH42 as the solvent. PSHs with more SBMA moiety than
HCPM units (PSH68 and PSH94) were diluted with trifluoroethanol (TFE). For
antibacterial test, 5 wt% of polymer solutions was prepared and drop casted onto
the silicon wafers and dried in vacuum overnight. Spin-coating and drop-casting
solutions contained 0.1 and 0.5 wt% of photoinitiator, 2-hydroxy-2-
methylpropiophenone respectively. The PSH coated substrates were irradiated
with 21700 mW/cm2 UV light (B-100APultraviolet lamp, UVP Inc., USA) at a
distance of 5 cm for 3 hours in air at room temperature to prepare PSH#C films.
11
2.6. Antifouling test
Antifouling test against bacteria was carried out with confocal laser scanning
microscope (CLSM, Eclipse 90i, Nikon, Japan) and Escherichia coli (E.coli;
ATCC 8739) was used as a representative. E.coli solution of a concentration of
107 CFU/mL was used for the assay. A 5 mL of the bacteria solution was dropped
on a 6-well plate containing silicon wafers (1.5 cm x 1.5 cm) coated with PSHs.
The plate was then shaken at 110 rpm inside a shaking incubator. After shaken for
24 hours at 25 oC, the samples were taken off from the 6-well plate, washed with
PBS solution thrice to remove loosely attached cells, and then placed on petri
dishes. A 200 μL of dye mixture of a PI (0.1 mmol/L in DIW) and SYTO9 (0.02
mmol/L in DIW) was dropped on the surfaces of the PSH#C films and the
samples were placed under dark condition for 1 hour. SYTO9 stains the live cells
green, whereas PI penetrates damaged cells and stains the cells red. After rinsing
for removal of the unbound dyes, stained bacteria cells attached from the surfaces
12
were observed through the microscope using 60X lens.
For measuring antifouling property of the samples against protein, BSA (Bovine
serum albumin) was used. BSA-fluorescence isothiocyanate (BSA-FITC, Sigma-
Aldrich) was dissolved in PBS to prepare a 0.01 % (w/v) solution. A 200 μL of
solution was poured on the samples. The samples were then incubated in a
humidified atmosphere of 5 % (v/v) CO2 and 95 % (v/v) O2 at 37 ºC for 30 min.
Finally, each sample was rinsed using the PBS three times, and then dried for 30
min at 37 ºC. After drying, BSAs attached to the PSH#C films were examined
using a fluorescence microscope (IX71 inverted microscope, Olympus, Japan).
2.7. Anti-HDF fouling and cytotoxicity test
Human dermal fibroblasts (HDFs) were purchased from Lonza (Walkersville,
MD). HDFs were cultured in a growth medium consisting of high-glucose
Dulbecco’s Modified Eagle’s Medium (Gibco BRL, USA) supplemented with 10 %
(v/v) fetal bovine serum (FBS, Gibco BRL, USA) and 1 % (v/v)
13
penicillin/streptomycin (Gibco BRL, USA). The HDFs were seeded (2 × 103
cells/cm2) on coverslips coated with PMMA and PSH#C films, and cultured in a 6
well-plates (Corning, USA).
Mitochondrial metabolic activity of the HDFs was determined by the 3-[4,5-
dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. A 200 μL of
MTT solution (5 mg/mL in PBS, Sigma-Aldrich) was added to the 6 well-plates
containing the PSH#C films and incubated for 4 hours at 37 ºC, then replaced with
dimethyl sulfoxide (DMSO, Sigma-Aldrich) to dissolve the formazan crystals.
The plates were placed in a humidified atmosphere of 5 % (v/v) CO2 and 95 %
(v/v) O2 at 37 ºC for 30 min to dissolve formazan completely. An absorbance at
570 nm was measured using a spectrophotometer. The HDFs cultured on the
PMMA film were served as a control group to demonstrate the effect of the
PSH#C films on cell viabilities. The morphology of the HDFs on the PSH#C
films was measured by a light microscope (IX71, Olympus, Tokyo, Japan).
Live and dead cells were detected with fluorescence microscopy after staining the
films with fluorescein diacetate (FDA, Sigma-Aldrich) and ethidium bromide (EB,
14
Sigma-Aldrich) at day 2 and 4 after the HDF seeding. FDA/EB solution was
prepared by mixing FDA solution (5 mg /mL in acetone) and EB solution (10
mg/mL in PBS) at final concentration 5 μg/mL and 10 μg/mL in PBS, respectively.
The PSH#C films cultured in the 6 well-plates were incubated in the FDA/EB
solution for 5 min at 37 ºC and then washed twice in PBS. Dead cells were stained
red due to a nuclear permeability of the EB. Viable cells capable of converting the
non-fluorescent FDA into fluorescent were stained green. After staining, the films
were examined using a fluorescence microscope (IX71 inverted microscope,
Olympus, Japan).
2.8. Antibacterial test
Antibacterial property of the PSH#C films was confirmed through film
attachment test. E.coli, a gram-negative type bacteria, was chosen for the assay.
To prepare the bacteria suspension, E.coli was grown in the corresponding broth
solutions at 37 °C for 18 hours. After culturing the bacteria, one of the colonies
15
was raised using platinum loop, put in a 30 mL of nutrient broth solution, and
grown with shaking at 37 oC for 18 hours. After washing three times with PBS
solution, the bacteria solution was diluted in PBS to make 106 colony forming unit
(CFU)/mL. A concentration of the bacteria suspension was estimated using
ultraviolet-visible (UV-Vis) spectrometer by measuring an absorbance of the cell
dispersions at 600 nm and referenced to a standard calibration curve. An optical
density (OD) value of 1.0 at 600 nm is approximately equivalent to 109 CFU/mL.
To evaluate the antibacterial characteristic of the PSH#C films, 0.1 mL of the
bacteria suspension was dropped on the PSH#C film surfaces (2 cm x 2 cm)
placed on a petri dish, and OHP film having a same size with the PSH#C films
covered the surface for full contact between the bacteria solution and the surface
at 25 °C. After 24 hours, the OHP film was peeled off and 0.9 mL PBS solution
was added into the petri dish that contained the sample. By shaking the petri
dishes strongly, the bacteria present on the substrates could be detached from the
surfaces and were transferred to micro tubes. The bacteria solutions were serially
diluted and 0.1ml of the diluent was spread on the agar medium. After 18 hours at
16
37 oC, remaining colonies were counted and each assay was repeated thrice. The
rate of bacterial inhibition was calculated using following equation:
Bacterial inhibition rate (%) =100 X (No- Ni)/No
where No is bacterial CFU of blank and Ni is bacterial CFU of tested samples.
2.9. Characterization
The chemical structures of monomer and polymers were confirmed by 1H NMR
spectroscopy (ZEOL LNM-LA 300, 300 MHz) using CDCl3 (Cambridge Isotope
Laboratories) as a NMR solvent and tetramethylsilane (TMS) as an internal
standard at a room temperature. The molecular weight (Mn and Mw) and
polydispersity index (PDI) of the polymers were characterized by gel permeation
chromatography (GPC). Relative molecular weight measurements were performed
using a Waters 515 HPLC pump equipped with three columns including PL gel
5.0 μm guard, MIXED-C, and MIXED-D from Polymer Laboratories at 35 oC in
series with a Viscotec LR125 laser refractometer. The system with a refractive
17
index (RI) detector was calibrated using polymethyl methacrylate (PMMA)
standards from Polymer Laboratories. HPLC grade tetrahydrofuran (THF, J. T.
Baker) was used as an eluent at a flow rate of 1.0 mL/min at 35 oC. The results
were measured using the Omnisec software. The element contents were calculated
for carbon, hydrogen, nitrogen and sulfur by an element analyzer, TruSpec®
Micro (Leco, USA). Thermal stability of the polymers was analyzed by a thermal
gravimetric analysis (TGA) using TA Instruments TGA Q-500 under nitrogen (N2)
atmosphere. The samples were heated to 120 oC with ramping rate of 10 oC/min
for removing remaining moisture and solvent, stayed for 5 minutes at 120 oC and
then heated to 700 oC with 10 oC/min heating rate. Thermal transition state of
polymers was analyzed by differential scanning calorimetry (DSC) with TA
Instruments DSC-Q1000 under N2 atmosphere. Samples weighing 3~7 mg were
encapsulated in sealed aluminum pans. The samples except PSH68 and PSH94
were heated to 120 oC at a heating rate of 10 oC/min and stayed for 5 minutes at
120 oC. The polymers were quenched to -50 oC with a cooling rate of 10 oC/min
and waited for 5 minutes at -50 oC, followed by a second heating scan from -50 oC
18
to 250 oC at a ramping rate of 10 oC/min. In case of the PSH68 and PSH94, the
same condition was applied except first heating scan range from -50 to 250 oC.
Fourier transform-infrared (FT-IR) spectra were recorded on Nicolet 6700
spectrophotometer (Thermo Scientific, USA) using Attenuated Total Reflectance
(ATR) equipment (FT-IR/ATR). The surface composition of the PSH#C films was
measured by X-ray photoelectron spectroscopy (XPS, Kratos Axis-HSI) using Mg
Kα (1254.0 eV) as the irradiation source. Survey spectra were collected over a
range of 0~1200 eV. The static contact angles (CA) of deionized water (DIW) on
the surfaces of the PSH#C films were measured at room temperature and ambient
relative humidity using a Krüss DSA 100 contact angle analyzer interfaced to drop
shape analysis software. The contact angles were measured by dropping a 4 μL of
the DIW onto the PSH#C films. The contact angle values for each sample were
measured more than five times on three independently prepared samples and the
average values were used as the data. Ultraviolet-visible (UV-Vis) spectra were
obtained by Agilent 8453 UV-Visible Spectrometer at room temperature.
19
3. Result and Discussion
3.1. Synthesis of 2-hydroxy-3-cardanylpropyl methacrylate (HCPM)
A methacrylate group was introduced into the plant-derived cardanol for
synthesis of a polymer with antibacterial property. Cardanol reacted with glycidyl
methacrylate in a presence of a basic catalyst, KOH (Scheme 1(a)).56 As a result,
2-hydroxy-3-cardanylpropyl methacrylate (HCPM) was synthesized through OH
group in the cardanol attacked epoxide ring of the glycidyl methacrylate. The
chemical structure of the HCPM could be confirmed by 1H NMR as shown in Fig.
1(a).
3.2. Synthesis of copolymers with DMAEMA and HCPM moieties
A series of PDH# (where # is the mol % of the DMAEMA moiety in the polymer)
were synthesized via free radical polymerization using DMAEMA and HCPM as
co-monomers and AIBN as an initiator (Scheme 1(b)). The DMAEMA was used
20
to introduce zwitterionic moiety into the polymer via betainisation using 1,3-
propane sultone. The chemical structures of PDHs were confirmed via 1H NMR.
In Fig. 1(b), (c) and (d), sharp peaks at 5.61 and 6.15 ppm from methacrylate
protons in the HCPM were disappeared and all of the corresponding peaks of
21
Scheme 1. Synthesis of (a) 2-hydroxy-3-cardanolpropyl methacrylate (HCPM), (b)
poly(DMAEMA-co-HCPM) (PDMs), and (c) poly(SBMA-co-HCPM) (PSHs).
22
23
Fig. 1 1H NMR spectra of (a) HCPM, (b) PDMAEMA, (c) PHCPM(PSH0) and (d)
PSH24.
PHCPM and PDMAEMA were appeared. Therefore, successful copolymerization
24
could be confirmed. Table 1. shows the results of the synthesis of PDHs from
different feed molar ratio of the DMAEMA and HCPM. The content of
DMAEMA in PDH was calculated from the 1H NMR data as follows:
DMAEMA content in PDH# (mol %)
= Id X feeding mole % of HCPM monomer/6,
where Id is the integral of the d proton peak in Fig. 1(d). The number average
molecular weight (Mn) of the PDHs analyzed by GPC equipped with a refractive
index detector was ranged between 8,800 and 19,800 as shown in Table 1.
25
Table 1. Synthesis results of the PDH#s from different co-monomers feeding
ratios.
Composition (DMAEMA : HCPM)
Samples Feed (%)
(mol : mol)
In polymera (%)
(mol : mol) Mnb (x10-3) PDIb
PDH0 0 : 100 0 :100 17.8 1.8
PDH21 20 : 80 21 : 79 10.7 2.0
PDH42 40 : 60 42 : 58 19.8 1.8
PDH68 70 : 30 68 : 32 8.8 2.5
PDH94 90 : 10 94 : 6 12.8 2.9
a Determined by H1-NMR. b Determined by GPC using refractive index (RI) detector and calibrated with linear polystyrene standards (THF).
3.3. Betainisation reaction of the PDHs
Zwitterionic moiety was introduced into the PDHs through betainisation with
1,3-propane sultone, and the resulting polymers were designated as PSH#, where
# is the molar compositional ratio of the sulfobetaine methacrylate (SBMA)
moiety in the polymers (Scheme 1(c)). A structure of the DMAEMA unit could be
changed into the zwitterionic form when an nitrogen of the DMAEMA moiety in
26
the PDH attacked carbon next to an oxygen in the 1,3-propane sultone.57 As
shown in Table 2., elemental compositions of the PSHs were analyzed by
elemental analysis (EA) and there was little difference in theoretical and
experimental weight % of carbon, nitrogen, sulfur, and hydrogen in PSHs within
2 % error, so it could be demonstrated that the 1,3-propane sultone reacted the
DMAEMA moiety in the PDHs successfully.
Table 2. Elemental analysis of the PSHs.
(Unit: wt %)
Element
PSH0 PSH21 PSH42 PSH68 PSH94
a b a b a b a b a b
C 88.6 88.8 85.7 86.1 82.4 81.7 76.8 77.5 68.7 71.1
N 0 0 0.9 0.9 1.9 1.4 3.7 3.3 6.3 5.5
S 0 0 2.1 1.8 4.4 5.8 8.5 8.0 14.3 12.2
H 11.4 11.2 11.3 11.2 11.2 11.1 11.0 11.1 10.7 11.2
Total 100 100 100 100 100 100 100 100 100 100
a Theoretical value. b Experimental value.
Thermal stability of the PSH series was confirmed by thermal gravimetric
27
analysis (TGA) as shown in Fig. 2. Thermal decomposition temperature at 10 %
weight loss of the PSHs increased with increase in a content of the HCPM moiety
in the PSH. This is attributed to a presence of benzene group in the HCPM unit
and formation of thermal cross-linked structures between unsaturated bonds in the
HCPM moiety.
Fig. 2 TGA thermograms of the PSHs under N2 atmosphere.
28
Fig. 3(a) shows differential scanning calorimeter (DSC) results of the PSHs. An
exothermic peak was appeared about 175 oC in the DSC results of the PSH0 and
PSH21 and this can be ascribed to a formation of the thermal cross-linked
structures between unsaturated bonds in the HCPM moieties during the DSC
heating scan. It is generally known that drying oils containing unsaturated double
bonds can be cross-linked under an air by auto-oxidation process.58 In the other
hand, a broad endothermic peak could be obtained around 150 oC in the DSC
graph from the PSH42, PSH68, and PSH96, and these peaks are attributed to a
dehydration of water bound to the SBMA moiety through electrostatic
interaction.59 In addition, the intensity of the endothermic peak increased as the
amount of the zwitterionic SBMA group interacting with water molecules
increased. As shown in Fig. 3(b), a glass transition temperature (Tg) value of the
PSHs decreased with increase in the amount of the HCPM moiety in the PSHs
because the long hydrocarbon chains in the HCPM act as a plasticizer, so they
inhibit close packing between the polymer chains. The Tg of the PSH68 and
29
PSH94 could be obtained after removing the moistures remained on the PSH
through the first heating in the DSC system because the endothermic peaks with
high intensity concealed the glass transition region. However, the Tg of the
PSH68 and PSH94 was more than twice compared to that of the PSH42 due to a
low portion of the HCPM moiety containing long hydrocarbon chains and the
formation of the thermally cross-linked structures.
30
Fig. 3 DSC thermograms of the PSHs under (a) 1st heating condition and (b) 1st
heating condition (PSH0, PSH21 and PSH42) and 2nd heating condition (PSH68
and PSH94).
3.4. Preparation of cross-linked PSH# (PSH#C) films
The PSHs were coated on the si-wafers and glass substrates using spin-coating
31
and drop-casting methods. Since the PSH contains both hydrophilic SBMA and
hydrophobic HCPM moieties, different types of solvents were used considering
the characteristics of the PSH series. The PSH0, PSH21, and PSH42 with more
hydrophobic moiety in the polymer were soluble in chloroform (CHCl3) and
tetrahydrofuran (THF). However, the PSH68 and PSH94 having more hydrophilic
group in the PSH were insoluble in aprotic polar solvents such as DMF, DMAc,
and DMSO because of the hydrophobic long hydrocarbon chains in HCPM
moiety existing in a low portion within the PSH. There is a report that
trifluoroethanol (TFE) or trifluoroacetic acid was used as a solvent for dissolving
amphiphilic copolymers containing both hydrophilic zwitterion and hydrophobic
moiety.60 Therefore, TFE was selected as the solvent for the PSH68 and PSH94.
Cross-linked PSH# (PSH#C) films were prepared via UV irradiation in order to
enhance the surface stability through the cross-linking reaction of the unsaturated
bonds in HCPM moieties. A degree of the cross-linking of the PSH#C films could
be monitored by an intensity change of C–H stretching vibration peak at 3010 cm-
1 from the unsaturated hydrocarbons in the HCPM using fourier transform
32
infrared spectroscopy/attenuated total reflection (FT-IR/ATR) (Fig. 4). The C-H
vibration peak intensity of the PSH0C film was decreased with the time and
ceased after 2 hours, indicating the fully cross-linked PSH film.
Fig. 4 FT-IR/ATR spectra of PSH0C film in the high frequency region before and
after UV irradiation for 1 and 2 hours.
33
3.5. Optical and chemical characteristics of the PSH#C films
Transmittance of the PSH#C films were measured using UV-Vis spectrometer in
a visible light region. As shown in Fig.5, all of the PSH#C films showed high
transparency similar to the bare glass substrate.
Fig. 5 UV-Vis spectra of cross-linked PSH (PSH#C) films on glass substrates.
Inset is photograph of the PSH#C films prepared by a spin coating method.
34
Elemental compositions of the PSH#C films were confirmed by X-ray
photoelectron spectroscopy (XPS) and summarized in Table 3. The atomic % of
nitrogen and sulfur increased as the amount of the SBMA moiety in the PSH
increased, and the experimental and theoretical values were similar. Moreover, the
atomic % ratio of nitrogen and sulfur in all of the PSH series was close to 1:1,
verifying that 1,3-propane sultone reacted with PDHs successfully via
betainisation.
Table 3. XPS analysis of the PSH#C films.
(Unit: atomic %)
Element
PSH0 PSH21 PSH42 PSH68 PSH94
a b a b a b a b a b
C 87.5 81.2 84.1 79.7 79.9 77.3 73.7 72.5 63.8 67.6
O 12.5 18.8 14.5 18.9 16.9 20.1 20.5 21.4 26.2 24.0
N 0 0 0.7 0.9 1.6 1.4 2.9 3.2 5.0 4.3
S 0 0 0.7 0.5 1.6 1.3 2.9 2.8 5.0 4.1
Total 100 100 100 100 100 100 100 100 100 100
a Theoretical value. b Experimental value.
35
Fig. 6 is the results of a water static contact angle analysis of the PSH#C films.
The contact angle decreased with increase of the SBMA content in the PSH
because the SBMA containing both positive and negative charge from ammonium
and sulfonate groups respectively can attract water molecules via ion-dipole
interaction. Therefore, it can be expected that PSH containing more hydrophilic
SBMA moiety has the excellent antifouling properties since the anti-adhesion
characteristic of the zwitterionic polymers is attributed to the formation of the
hydration layer.
36
Fig. 6 Water static contact angles of the PSH#C films. Inset is photographic
results.
3.6. Antifouling properties of the PSH#C films
Antifouling characteristics of the PSH#C films were conducted using bacteria
and protein. Escherichia coli (E. coli) was chosen for the anti-adhesion analysis of
37
the films against bacteria. PMMA film was prepared for a positive reference since
PMMA has no antifouling and antibacterial properties. As shown in Fig. 7, all of
the cells observed on the PMMA film glowed a fluorescent green, indicating the
live cells. On the other hand, only dead cells stained by red dye were attached on
the PSH0C film due to the bactericidal HCPM moiety. A number of the cells
attached on the film decreased while the portion of the SBMA group in the PSH
increased and there were no bacteria on the surfaces in case of the PSH68C and
PSH94C film. This can be explained that the hydration layers formed by
interaction between SBMA units and the water molecules were developed more
densely on the PSH#C film as the content of the zwitterionic moiety increased, so
the bacteria accessing into the PSH#C films could not be fouled from the surface.
38
Fig. 7 Representative fluorescence microscope images of adhered E.coli onto the
PMMA and PSH#C films.
The results of the protein adsorption on the PSH#C films were shown in Fig. 8.
The color of the PMMA and PSH0C film was green, which indicated the
adsorption of the proteins, Bovine serum albumin (BSA). However, the number of
adsorbed proteins on the PSH21C was decreased drastically and there were no
BSAs on the PSH42C, PSH68C, and PSH94C films. From these results, it is
confirmed that the anti-protein property of the PSH is caused by the zwitterionic
39
moiety in PSH.
Fig. 8 Representative fluorescence microscope images of adhered BSA onto the
PMMA and PSH#C films (scale bar = 100 µm).
3.7. Antifouling and cytotoxic characteristics of the PSH#C films against
HDFs
Fig. 9 and 10 indicate the anti-adhesion property and cytotoxicity results of the
40
PSH#C films using Human dermal fibroblasts (HDFs). As shown in Fig. 9, the
number of HDFs attached on the PSH#C film decreased when a portion of the
SBMA moiety in PSH increased. In addition, in the day 4 data from the PMMA,
PSH0C, and PSH21C films (Fig. 9(b)), the amount of the cells on the films
increased compared to the day 2 results (Fig. 9(a)). However, there was little
difference in the number of the HDFs on the PSH42C, PSH68C, and PSH94C
films between day 2 and day 4 data. From these results, it can be expected that
PSHs with more hydrophilic SBMA moiety have enhanced anti-HDF fouling
properties because hydration layers preventing the attachment of the cells onto the
PSH#C film can be formed more densely. Moreover, it is concluded that the HDFs
proliferated by the fouled cells on the PSH#C films containing more zwitterionic
moieties are difficult to attach onto the surface stably due to the densely formed
hydration layers.
Fig. 10 shows the cytotoxicity of the PSH#C films using a qualitative assay of
live and dead cells, wherein live and dead cells are stained green and red,
respectively. Non-viable HDFs stained by a red dye, ethidium bromide (EB), were
41
not observed on all of the PSH#C films, confirming that PSHs have no cytotoxic
behavior to the human body.
42
43
44
Fig. 9 Representative optical microscope images of HDFs cultured for (a) 2 and
(b) 4 days on the surfaces of the PMMA and PSH#C films (scale bar = 100 µm).
Cell viability determined by MTT assay after (c) 2 and (d) 4 days.
45
46
Fig. 10 CLSM images presenting FDA/EB staining on HDFs cultured for (a) 2
and (b) 4 days on the PMMA and PSH#C films (scale bar = 100 µm).
3.8. Antibacterial properties of the PSH#C films
47
Antibacterial property analysis of the PSH#C films was conducted against E. coli
using a film-attachment method. However, PSH#C films prepared by spin-coating
method did not show the antibacterial property due to very low thickness of the
films. In the antibacterial tests, therefore, drop casting method was used for the
preparation of the PSH#C films because it is known that film thickness can affect
the bactericidal characteristic of the film. However, the PSH68C and PSH94C
films could not be used in the test because the films were broken. It can be
expected that the amount of the cross-linkable HCPM moiety in the PSH68 and
PSH94 is not enough to make the stable film. In addition, a low molecular weight
of the PSH series, ranging between 11,000 and 23,000, might cause the breakage
of the films. It is well-known that a polymer with high molecular weight is
susceptible to an intermolecular and intramolecular entanglement within the
polymer chains. So it can be assumed that the PSHs consisting of short molecular
chains have little chance of inter and intra chain entanglement, resulting the brittle
film.61 On the other hand, the PSH0C, PSH21C, and PSH42C film were stable
48
because there is a sufficient amount of the HCPM unit in the PSH to make the
stable film through the UV cross-linking process despite they have low molecular
weight. Fig. 11(a) and (b) show the bacterial inhibition efficiency of the PSH#C
films and all the films used in the test had an excellent antibacterial property, >
99 %. From these results, the effective antibacterial characteristic of the PSH#C
films due to the HCPM moiety was confirmed.
49
Fig. 11 (a) Photographs of the PMMA and PSH#C films via antibacterial test
against E.coli. (b) Bactericidal activity of the PMMA and PSH#C films.
4. Conclusion
PSH series with antifouling and antimicrobial properties were synthesized via
free radical polymerization of DMAEMA and HCPM, followed by betainisation
50
reaction. To confirm a feasibility of the PSHs as a coating material in anti-
biofouling applications, PSH films were fabricated through spin-coating and drop
casting methods. Cross-linked PSH# (PSH#C) films were prepared via UV cross-
linking between the double bonds within the HCPM to enhance a stability of the
films. The PSH#C films were transparent and had an excellent antibacterial
characteristic due to the HCPM group in the PSH. Moreover, PSH#C films
showed good antifouling effects against bacteria, protein, and HDFs with increase
of the hydrophilic SBMA moiety in the PSH, and all of the PSH#C films had non-
cytotoxicity toward the HDFs. Therefore, considering all of the aspects, film
stability, bacterial inhibition, adhesion-resistance, and cytotoxicity, it can be
suggested that PSH42 is very promising candidate for the anti-biofouling coating
material.
51
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59
문
항균 특 가진 공 합체 PSH#(# 공 합체에
재하는 mol %를 미)가 DMAEMA HCPM
를 용한 라 칼 합 1,3-propane sultone 용한 개
질 통해 합 었다.
합 한 PSH 시리 들 스핀코 드랍캐스 용하
여 에 코 한 후 UV 사 과 통해 가 가 형 필
름(PSH#C필름) 하 , 필름 특 들 하
다.
그 결과 PSH#C 필름 가시 역에 과도를 보
, 고 내에 친수 SBMA 비 가함에
라 물에 한 낮 각 나타내었다.
리 (E.coli) 단 질(BSA)를 용하여 PSH#C 필름
특 한 결과, 고 에 SBMA 많 재할수록
우수한 특 나타내는 결과를 얻 수 었다. 또한 든
PSH#C 필름 체 포(HDF)에 무해하 , 고 내에
60
특 현하는 SBMA 비 가함에 라 체
포에 해 도 착특 없는 결과를 확 할 수 었다.
PSH#C 필름 고 내에 재하는 HCPM로 해, 리
에 해 99 % 상 항균 특 나타내는 결과를 얻
수 었다.
라 PSH 항균 특 각각 SBMA HCPM에
한 것 확 할 수 었다. 특 , 항균 특 우
수하게 나타난 PSH42는 생물체로 한 생물막 형 지가 필
한 에 코 재로 용하게 쓰 수 것 로
상 다.
주 어: , 카다 , , 항균, 코 재
학 : 2014-20564