Catalyst free-click polymerization: A versatile method for the preparation of soybean oil based poly1,2,3-triazoles as coatings with efficient biocidal activity and excellent cytocompatibility

Article abstract of DOI:10.1016/j.polymer.2015.02.029

Click polymerization was applied for the preparation of soybean oil-based poly1,2,3-triazoles (PTAs). Azidated soybean oil (ASBO) was prepared and reacted with two different propyn-functional urethane monomers. Azidated derivatives of a quaternary pyridinium salt (APS) and Cloisite 30B (MMT-N₃) were also synthesized and tethered into the PTAs to control the physico-mechanical properties as well as biological activities. PTAs showed very good hardness (169-270 s^-1) and adhesion strength (0.65-1.65 MPa) to Aluminum plate as model substrate. Qualitative and quantitative assessments confirmed promising cytocompatibility of PTAs against fibroblast cells (86-98% viability). Meanwhile, introduction of either APS or MMT-N₃ in PTAs induced bactericidal and fungicidal activities against S. aureus, P. aeruginosa and C. albicans. Higher biocidal activity (up to 99% reduction) was detected when these modifiers were used mutually in coatings materials. This work provides a new insight for utilizing a biorenewable material for the preparation of high-performance coatings suitable for biomedical applications.

Full text of DOI:10.1016/j.polymer.2015.02.029

Accepted Manuscript
Catalyst free-click polymerization: a versatile method for the preparation of soybean
oil based poly1,2,3-triazoles as coatings with efficient biocidal activity and excellent
cytocompatibility
Hoshayer Gholami, Hamid Yeganeh, Reza Gharibi, Mehrdad Jalilian, Marziyeh
Sorayya
PII:
S0032-3861(15)00189-5
10.1016/j.polymer.2015.02.029
JPOL 17642
DOI:
Reference:
To appear in: Polymer
Received Date: 9 December 2014
Revised Date: 28 January 2015
Accepted Date: 17 February 2015
Please cite this article as: Gholami H, Yeganeh H, Gharibi R, Jalilian M, Sorayya M, Catalyst free-
click polymerization: a versatile method for the preparation of soybean oil based poly1,2,3-triazoles as
coatings with efficient biocidal activity and excellent cytocompatibility, Polymer (2015), doi: 10.1016/
j.polymer.2015.02.029.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Catalyst free-click polymerization: a versatile method for the
preparation of soybean oil based poly1,2,3-triazoles as coatings with
efficient biocidal activity and excellent cytocompatibility
Hoshayer Gholami, Hamid Yeganeh*1, Reza Gharibi, Mehrdad Jalilian and Marziyeh Sorayya
Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran, Iran
Abstract
Click polymerization was applied for the preparation of soybean oil-based poly1,2,3-triazoles
(PTAs). Azidated soybean oil (ASBO) was prepared and reacted with two different propyn-
functional urethane monomers. Azidated derivatives of a quaternary pyridinium salt (APS) and
Cloisite 30B (MMT-N3) were also synthesized and tethered into the PTAs to control the physico-
mechanical properties as well as biological activities. PTAs showed very good hardness (169-
270 s-1) and adhesion strength (0.65-1.65 MPa) to Aluminum plate as model substrate.
Qualitative and quantitative assessments confirmed promising cytocompatibility of PTAs against
fibroblast cells (86-98 % viability). Meanwhile, introduction of either APS or MMT-N3 in PTAs
induced bactericidal and fungicidal activities against S. aureus, P. aeruginosa and C. albicans.
Higher biocidal activity (up to 99% reduction) was detected when these modifiers were used
mutually in coatings materials. This work provides a new insight for utilizing a biorenewable
material for the preparation of high-performance coatings suitable for biomedical applications.
Keywords: Click polymerization, biocidal activity, biorenewable materials
Introduction
Bacterial and fungal contaminations remains as one of the most serious complications in several
areas, particularly in medical devices [1,2], water purification systems [3,4], hospital/dental
surgery equipments [5–7] and food packaging [8,9]. This trouble raised a serious global concern,
since it can greatly influence on the health and lifetime of people[10,11]. Coatings with biocidal
1
To whom all correspondence should be addressed.
Tel: +9821 48662447, Fax: +9821 44787023, e-mail: h.yeganeh@ippi.ac.ir
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activity offer an approach to limit the spread of microbial infections in many of the
aforementioned area. Because of versatility and diversity of methods available for the
preparation and application of polymeric coatings, this category of materials found special
position in this approach. Impregnation of biocidal additives into the polymeric coating
materials is the most frequent technique used to provide biocidal activity in coatings [12–14].
However, coatings made from this method show temporary biocidal activity. Gradual release of
active ingredients not only will deteriorate the biocidal performance of coatings, but also, the
released low molecular weight biocidals into the surrounding environment may cause human
toxicity and microbial resistance effects [15,16]. To endow permanent antimicrobial activity and
produce hygiene surfaces, preparation of polymeric coatings with tethered biocides and contact
killing power of harmful microorganisms is a more efficient approach. In this method, the release
of biocides into the environment is avoided and consequently the service time of coating can be
prolonged. As well, the biocidal efficiency/selectivity can be increased as a result of high local
concentration of biocides at the surface of materials [12,17–19]. Preparation of biocidal
polymeric coatings through reaction of reactive monomers and functional additive is considered
in the present work. To this end, a quaternary pyridinium salt with well-known extensive
biocidal activity [20–22] was introduced into an alkyl azide molecule. Cloisite 30B (a quaternary
ammonium salt modified clay) was also functionalized with azide group[23] and used as a
reactive additive during polymerization reaction for enhancing both the physico-mechanical
properties [24–26] and biocidal activity [27–29] of final polymers. Poly1,2,3-triazole (PTA) was
selected as the structural framework of coating materials, since it has been proved that this
category of materials endow excellent coating properties such as high thermal stability, good
mechanical properties, hydrolysis and redox resistance, high adhesion to metals surface and
antibacterial activity [30–36]. Presence of 1,2,3-triazole five member ring with high dipole
moment and possibility to participate in hydrogen bonding type interaction is responsible for the
mentioned properties of PTA based coatings. Preparation of PTA mainly carries out by alkyne-
azide Huisgen cycloaddition reaction. This reaction is a well-known example of a class of
chemical reactions called “click reactions” [37,38]. Click reactions are characterized by facile
experimental set-up and minimal synthetic work-up, high quantitative yields and tolerance
towards the use of different functional groups. These reactions are also relatively insensitive to
the solvent system and can be easily performed even in aqueous solvent under aerobic condition.
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Click reactions are very good tools for polymer synthesis, if multifunctional azides and alkynes
are employed as starting materials [39–41]. As a multi functional azide monomer, azidated
soybean oil (ASBO) was selected in the present work. ASBO is an example of vegetable oil
derived monomers that found extensive applications for the preparation of polymers with many
environmental benefits [42–45]. Utilizing the power of click chemistry for functionalization and
polymerization of vegetable oils has attracted the attention of polymer chemist [46]. Application
of azidated and alkynated vegetable oils (castor, canola, corn, soybean and linseed oils) for the
preparation of green PTAs under Cu(I)-catalyzed polymerization at room temperature or in a
º
catalyst-free and solvent free environment at 100 C has been recently reported by Shah and
coworkers [47]. We have also utilized the click reaction for creating 1,2,3-triazole functions with
and without quaternary ammonium salts moieties in the backbone of soybean oil [48]. The
polyurethanes prepared from these functional oils have subjected to several physical, chemical
and mechanical analyses. To explore the possible application of these polymers as coatings for
medical devices, the biological activities of these polymers against mammalian, bacterial and
fungal cells were evaluated and correlated to the polymers structure.
In continuation of our ongoing research on the preparation of vegetable oil based polymers with
tailor made biological activity, hereby we report our findings regarding the synthesis and
assessments of PTAs made from ASBO trough click polymerization reaction. Two bi and tri
propyn-terminated urethane monomers were also synthesized through the reaction of propargyl
alcohol with suitable isocyanate monomers. An azide functional quaternary pyridinium salt and
azidated organically modified clay were synthesized and combined with aforementioned
monomers to control the physico-mechanical properties as well as biological activities of
coatings. All materials were characterized by conventional spectroscopic methods and based on
measured properties their possible application as coating of medical devices, implants and drug
delivery tools was evaluated.
Experimental
Materials
Epoxidized soybean oil (ESBO, PATSTAB 901) with a number-average molecular weight of
about 1000 and epoxy content of 3.47 mmol epoxy/g was purchased from PATCHEM (Sharjah,
UAE) and used as received. Sodium azide (NaN3), benzenesulfonyl chloride, 1-methylimidazole,
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propargyl alcohol (PrAl), pyridine, 1,4-dibromobutane, tetrabutylammonium hydrogen sulfate,
dibutyltin dilaurate (DBTDL), Tryptic Soy, Agar-agar Broth, NaCl, soybean peptone, casein
peptone, soybean peptone, disodium hydrogen phosphate, glucose and N, N-dimethylformamide
(DMF) all from Merck were used as received. Isophorone diisocyanate (IPDI) from Merck was
purified via vacuum distillation. Tetrahydrofuran (THF) was distilled over sodium wire.
Triphenylmethane 4,4',4''-triisocyanate in ethyl acetate (Desmodure RE) was purchased from
Bayer and used as received. Cloisite 30B was supplied from Southern Clay Products.
Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Candida
albicans (ATCC 10231) were provided by Iranian Research Organization for Science and
Technology (Tehran, Iran).
Synthesis of ASBO
ASBO was prepared according to procedure reported in our previous publication [48]. Briefly,
ESBO (24.0 g, 83.3 mmol epoxy), NaN3 (8.33 g, 128.2 mmol), 1-methylimidazolium
tetrafluoroborate (2.025 g 11.9 mmol), and DMF (20 g) were added into a round bottomed flask
equipped with a condenser, magnetic stirrer, and oil bath. The reaction mixture was stirred at
95 °C for 48 h. Then, the flask content was dissolved in ethyl acetate (100 ml) after cooling to
room temperature. The solution was successively washed with distilled water (100 ml) and
saturated sodium chloride solution (100 ml) to remove the catalyst residue and unreacted sodium
azide. The organic layer was dried over magnesium sulfate and the solvent was removed under
vacuum at 40 °C. ASBO was obtained as a dark-brown and viscous liquid at 90% yield.
Synthesis of N-(4-azidobutyl) pyridinium bromide (APS)
Firstly, N-(4-bromobutyl) pyridinium bromide was synthesized according to the procedure
described previously [49]. Briefly, a three-necked round bottomed flask equipped with a
magnetic stirrer, condenser, dropping funnel and a nitrogen inlet was charged with pyridine (1.60
g, 0.02 mol) and dry benzene (10 ml). A solution of 1, 4-dibromobutane (43.18 g, 0.20 mol) in
dry benzene (10 ml) was dropped to the flask. The reaction mixture was refluxed for 6 h. The
stirring was continued at room temperature overnight. The precipitated gummy solid was
filtered, washed with cold benzene and then dried under vacuum at 60 °C overnight (99% yield).
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Then, a two-necked round bottomed flask equipped with a magnetic stirrer, condenser, and
thermometer was charged with a solution of N-(4-bromobutyl) pyridinium bromide (2.95 g, 0.01
mol), NaN3 (1.30 g, 0.02 mol) and tetrabutylammonium hydrogen sulfate (0.034 g, 0.0001 mol)
in water (30 ml). The temperature was raised to 80 °C and the reaction was continued overnight
(about 15 h). The reaction mixture was subjected to reduced pressure. The resulting crystals at
85% yield were directly used without further purification.
Synthesis of bi-propyn-terminated urethane monomer (BPUM)
A three-necked round bottomed flask equipped with a magnetic stirrer, condenser, and a nitrogen
inlet was charged with PrAl (3.03 g, 0.054 mol), IPDI (5.00 g, 0.023 mol), one drop of DBTDL
and dried THF (30 ml). The reaction mixture was stirred at room temperature for 0.5 h, and then
temperature was increased to 80 °C. The reaction was continued until no free NCO peak at 2270
cm−1 was detected in the FTIR spectrum of sample collected from reaction mixture. The product
at 90% yield was obtained after vacuum evaporation of solvent and excess amount of PrAl at 80
°C overnight.
Synthesis of tri-propyn-terminated urethane monomer (TPUM)
A three-necked round bottomed flask equipped with a magnetic stirrer, condenser, dropping
funnel and a nitrogen inlet was charged with PrAl (3.03 g, 0.054 mol). Temperature was
increased to 60 °C and Desmodure-RE (18.51 g, 0.013 mol) was slowly dropped into the flask
during 30 min. The reaction was continued until no free NCO peak at 2270 cm−1 was detected in
the FTIR spectrum of sample collected from reaction mixture. The product at 94% yield was
obtained after vacuum evaporation of solvent and excess amount of PrAl at 80 °C overnight.
Synthesis of azide-functionalized Montmorillonite (MMT-N3)
MMT-N3 was prepared according to previously reported procedure with some modification [23].
Cloisite 30B (2.00 g, 0.00236 mol OH content), triethylamine (1.19 g, 0.0265 mol) and THF (80
ml) were added to a 250 ml two-necked round-bottomed flask equipped with a magnetic stirrer,
dropping funnel and thermometer. The mixture was cooled to 0 °C in an ice bath. While stirring,
benzenesulfonyl chloride (2.08 g, 0.0118 mol) was slowly dropped into the reaction mixture
during 30 min. Then, the reaction mixture was allowed to heat up to room temperature and
stirred overnight. Solvent was removed, and then ethanol (80 ml) was added to the reaction
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mixture. Sodium azide (0.77 g, 0.0118 mol) was added, and the reaction mixture was refluxed
overnight. After cooling to room temperature and removing the solvent, diethyl ether (80 ml)
was added to the reaction mixture. The mixture was washed three times with a saturated NaCl
aqueous solution. The crude product was obtained after evaporation of diethyl ether. It was
dispersed in water and centrifuged. This process (dispersion of functional clay in water and
centrifugation) was repeated three times and then the functionalized clay vacuum dried. MMT-
N3 was obtained at 90%yield.
Preparation of poly1,2,3-triazoles (PTAs)
Appropriate amounts of ASBO and the propyn-terminated monomer (1:1 molar ratio of -N3:
HC≡C- functions) were dissolved in DMF and solid content was fixed at 50 wt %. The mixture
was stirred vigorously at 70 °C for 2 h. Free standing films of PTAs with final thickness of about
0.5 mm were prepared by casting of the reaction mixture in a Teflon mould (10 × 10 × 0.2 cm3)
and thermally treated at 100 °C for 24 h. For evaluating the adhesion strength, hardness and
contact angle of PTAs, the reaction mixture was poured on the surface of a surface treated
Aluminium plate (Aluminium alloy 1050, 10 × 5 cm2) and spread with a doctor blade apparatus
(200 ꢀm). The plate was placed into an oven and thermally treated at 100 °C for 24 h. For those
formulations containing APS the solution of this material in few drops of water was added to
reaction mixture, and for those formulations containing MMT-N3 this additive was added to the
reaction mixture at the beginning of reaction.
The surface treatment procedure of Aluminium plate included washing of the plate with acetone,
sinking it in NaOH solution (0.1 M) for 2 h, neutralizing it with HCl solution (0.01 M) and
washing with distilled water.
The quantities of ingredients for each formulation are given in Table 1.
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Table 1.Different formulations of poly1,2,3-triazoles
Sample
code
ASBO
(g)
APS
(g)
MMT-N3
(g)
Curing agent
type
Curing agent
(g)
Gel
content
PTA1
PTA2
PTA3
PTA4
PTA5
PTA6
1
1
0
0
2
2
0
2
0
0
TPUM
BPUM
TPUM
BPUM
TPUM
TPUM
0.49
0.46
0.55
0.51
0.51
0. 48
98.4 ± 1.5
94.6 ± 2.2
92.0 ± 1.9
88.3 ± 1.7
97.1 ± 0.9
95.5 ± 0.2
0.8
0.8
1
0
0
0.05
0.05
0.8
Measurements
1H NMR spectra were recorded on a 300 MHz Bruker instrument (Germany) using D2O and
(CD3)2SO as solvents. FTIR spectra were obtained using a Bruker IFS 48 instruments. All
spectra were obtained under air as a function of time with 16 scans at a resolution of 4 cm-1 and a
spectral range of 500–4000 cm-1. Dynamic mechanical thermal analysis (DMA) was carried out
with a Tritec 2000 DMA instrument in torsion mode, temperature range −100 to 200 °C, heating
rate of 3 °C/min and a frequency of 1 Hz. The values of storage modulus, loss modulus and tan δ
versus temperature were recorded for each sample.
Thermogravimetric analysis (TGA) was carried out on a Mettler-Toledo instrument (model
TGA/DSC 1, Switzerland) from 25 to 600 °C at a heat rate of 10 °C/min under nitrogen
atmosphere. The bromide and silicon atoms contents and their distribution maps were measured
by a scanning electron microscope (SEM, Tescan, Vega II, Czech) equipped with an energy
dispersive X-ray analyzer system (EDX, Oxford Instrument, INCA, England). The X-ray
diffraction (XRD) pattern of the samples were recorded in a Siemens X-ray diffractometer
(D5000 GERMANY) at crystal monochromated Cu Ka radiation in the angular range 2–15 (2θ)
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at 35 kV operating voltage and 25 mA current. Transmission electronic microscopy (TEM)
micrographs were obtained with a Philips EM 208 apparatus using an acceleration voltage of 100
kV. Ultrathin sections (ca. 100 nm thick) were cut at -120 ºC from 0.5 mm thick films obtained
from casting and thermal treatment of reaction mixture in a Teflon mold using a Leica EM UC7
ultramicrotome equipped with a diamond knife. No selective staining was required due to large
difference in electron density between silicate and poly1,2,3-triazole matrix.
Hardness of prepared coatings was determined by a pendulum hardness tester (Elcometer, model
3034, England) on persoz mode according to ASTM D4366 test method. Adhesion strength of
coatings on an aluminium substrate was also studied by a pull-off adhesion tester (Erichsen
Testing Equipments, model 525, USA) according to ASTM D4541 test method.
To evaluate the network formation, gel content of coatings was determined. The vacuum dried
samples were weighed accurately and then extracted by THF in a Soxhlet extractor for 24 h. The
insoluble part was dried at 50 °C and weighed. The gel content was defined as follows:
ꢇꢈ
ꢀꢁꢂ ꢃꢄꢅꢆꢁꢅꢆ% =
× 100
ꢇ
ꢉ
Where Wi and Wd designate the initial weight and weight of dried membrane after extraction.
Surface hydrophilicity of membranes was determined by measuring the water droplet contact
angle at six different positions of membrane surface for each sample. The contact angle was
determined via running Image J 1.44p software on the digital pictures taken from the interfaces
of membranes and droplets. The values reported are an average of six measurements.
The bulk hydrophilicity of samples was evaluated based on equilibrium water absorption (EWA)
of samples in distilled water. For this purpose, the samples were completely dried at 70–80 °C,
accurately weighed and soaked in distilled water at room temperature until the equilibrium
swelling was attained (about 48 h). The weight of swelled sample was determined after being
gently wiped with filter paper to remove the surface liquid. EWA (%) was determined using the
following equation
ꢌ
ꢍ
ꢇ − ꢇꢈ
ꢎ
ꢌ ꢍ
ꢊꢇꢋ % =
× 100
ꢇ
ꢈ
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Where Wd and Ws designate the weight of dry and swelled samples, respectively.
Cross-link density (νc) of the coatings was estimated from their storage modulus in the rubbery
region by applying an equation from the statistical theory of rubber elasticity.
ꢀ′ = ΦνcRT
Cytocompatibility Assays
Cytocompatibility of coatings were studied by either microscopicinvestigation of fibroblast cells
morphology after direct contact with samples or tetrazolium dye-based colorimetric assay (MTT
assay) according procedures reported in our previously published articles [48]. Samples were
initially washed and extracted with ethanol/water mixture (70:30 v/v) for 24 h to remove all non-
bonded compounds. The percentage of relative cell viability was calculated according to
following equation:
ꢕꢖꢗꢘꢙꢚꢛꢜ − ꢕꢖꢚꢝꢗꢉꢞꢉꢟꢜ ꢠꢝꢡꢞꢢꢝꢛ
ꢏꢁꢂꢂ ꢐꢑꢒꢓꢂꢑꢆꢔ % =
× 100
ꢕꢖꢡꢜꢣꢘꢞꢉꢟꢜ ꢠꢝꢡꢞꢢꢝꢛ
, where OD designates the optical density
Biocidal Assays
The biocidal activity was determined based on ASTM E 2180 – 07. 18 h cultures prepared from
three microorganisms including Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa
(ATCC 15442) and Candida albicans (ATCC 10231) in tryptic soy broth. Microbial broth
cultures were adjusted to 3×108 cells/ml with a spectrophotometer. 1 ml of these cultures was
inoculated into Erlenmeyer flasks containing 100 ml molten agar slurry (0.3 g Agar-agar, 0.85 g
NaCl, 100 ml water). A thin layer of the inoculated agar slurry (100 µl) is pipetted onto the test
and untreated control materials (triplicate samples). After the specified contact time (24 h
commonly used), surviving microorganisms are recovered via elution of the agar slurry
inoculums from the test substrate into neutralizing broth and extracted via vortexing that provide
complete removal of the inoculums from the test polymers. Serial dilutions were done, and then
spread plates were made of each dilution. Agar plates and dilution broths are incubated for 48±2
h at 37 ºC as optimal temperature for test organisms. Microbial colonies from each dilution series
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were counted and recorded. The percentage of microorganism reduction was calculated
according to the following equation:
logꢦꢧ ꢨꢦ +logꢦꢧ ꢨꢩ + logꢦꢧ ꢨꢪ
ꢀꢁꢄꢤꢁꢆꢥꢑꢃ ꢤꢁꢒꢅ =
3
ꢌ
ꢍ
ꢒ − ꢓ × 100
ꢫꢁꢬꢭꢃꢆꢑꢄꢅ% =
ꢒ
,where x designates number of organisms recovered from the incubation period control or
incubation period treated samples, a designates the antilog of the geometric mean of organisms
recovered from the incubation period control samples and b designates the antilog of the
geometric mean of organisms recovered from the incubation period treated samples.
Statistical analysis
Statistical analyses were performed via PASW Statistics program package, version 18 (SPSS,
Chicago, IL). Comparison of obtained data for different samples was performed with one-way
analysis of variance with Tukey post hoc tests. The significance level was set at p ≤0.05.
Results and discussion
Synthesis and characterization of clickable starting materials, and poly1,2,3-triazoles
Selection of chemical framework and the sequences of reactions need for the utilization of
natural resource based raw materials have prime importance for reaching to the materials with
appropriate properties in comparison to what can be attained from common petroleum derived
raw materials. To this end, click polymerization method, azidated soybean oil, clickable clay and
biocidal additives were selected as main elements for fulfilling the aims of the present work.
Azidated soybean oil made from epoxidized soybean oil. For this purpose, the latter material was
subjected to the reaction with sodium azide in the presence of 1-methylimidazolium
tetrafluoroborate as catalyst. The synthesize, characterization and optimization of reaction
condition were fully described in our previous article [48].
The propyn-functional materials were easily prepared through the reaction of propargyl alcohol
with isophorone diisocyanate as a bifunctional aliphatic and Desmodure RE as a trifunctional
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aromatic isocyanate monomers. These molecules containing urethane linkages, therefore, it was
expected that hydrogen bonding type interactions of these functions with each other and with
1,2,3-triazole rings cause an increase in overall physico-mechanical properties of final networks
[48]. As well, urethane bonds provide good interaction with metallic substrates [48], and
therefore, higher adhesion was expected. The identity of BPUM and TPUM was confirmed by
evaluation of their FTIR and 1HNMR spectra (Figure 1 and 2). In none of FTIR spectra, the peak
related to free NCO groups at 2230 cm-1 were detected that revealed complete reaction of PrAl
with NCO groups. The broad band at about 3300 cm-1, was related to the stretching vibration of
urethane N-H bond. The urethane carbonyl (NH-CO-O-) was detected as a strong peak at about
1710 cm-1. The peak observed at about 1530 cm-1 was attributed to the C-N stretching, combined
with N-H out of the plane bending vibration of urethane linkages. The stretching vibration of
propyn bond (CH≡C-CH2-) was appeared at 2126 cm-1 and also stretching vibration of the
terminal C–H bond of propyn group was detected at about 3100 cm-1.
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Figure 1. FTIR spectra of (A) TPUM and (B) BPUM
1
The HNMR spectra of these two compounds (BPUM and TPUM) were also in accordance to
their proposed structures. As it is clear from Figure 2, the peaks related to HC≡C- function was
appeared at about 3.2 ppm. The methylene group bond to urethane oxygen atom (NHCOO-CH2-)
was detected at about 4.7 ppm. The presence of this peak confirmed the reaction of propargyl
alcohol with isocyante group. For TPUM monomer the central methine group (CH) was noticed
as a peak at 5.3 ppm and aromatic protons of benzene rings were appeared as peaks at 6.9 and
7.8 ppm. For BPUM monomer, the methyl groups of IPDI backbone were detected at about 0.9
ppm and methylene groups of cyclohexane ring were noticed in the range of 1.1-1.7 ppm. The
methylene and methine groups attached to urethane nitrogen atom were also appeared at 2.4-2.7
and 3.2 ppm, respectively.
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Figure 2. 1HNMRspectra of TPUM and BPUM
APS was used to induce biocidal activity in poly1,2,3triazole coatings. This compound belongs
to the general category of quaternary ammonium salts (QAS) [12]. QAS found important
position among disinfectants due to their relatively low toxicity to human and animal cells and
their good activity against wide spread types of microorganisms. APS was prepared through
nucleophilic substitution reaction of large excess amount of 1,4-dibromobutane with pyridine in
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benzene as solvent. The intermediate mono-substituted salt, N-(4-bromobutyl) pyridinium
bromide was readily precipitated out of solvent and then subsequently subjected to another
nucleophilic substitution reaction with sodium azide to prepare APS molecule.
1
The structure of APS was elucidated by FTIR and HNMR spectroscopy. In FTIR spectrum of
APS (Figure 3A), the azide group was detected as a strong peak at about 2100 cm-1. The small
shoulder at 2030 cm-1 was attributed to N3 counter anion produced through partial exchange of
Br anion located on pyridinium salt moiety. The aromatic pyridine ring was detected at about
1630 cm-1. The peak at 1488 cm-1 was attributed to C-H bending vibration of CH2-N+ moiety
and the stretching vibrations of aliphatic CH2 groups were also detected at 2868 and 2940 cm-1.
1
The HNMR spectrum of prepared APS is showed in Figure 3B. The peaks related to aromatic
C-H bonds of pyridine ring were appeared in the range of 7.9-8.7 ppm. The peaks related to the
methylene groups attached to azide and pyridinium moieties were appeared at 3.3 and 4.5 ppm
respectively. The central methylene groups were detected at 1.5 and 2.0 ppm. According to
1
integration values of main peaks appeared in the HNMR spectrum of APS, this compound was
at 96% purity.
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Figure 3. FTIR (A) and 1HNMR (B) spectra of APS
Azidation of organic modifier (methyl, tallow, bis-2-hydroxyethyl, quaternary ammonium
chloride) incorporated into the clay gallery (Cloisite 30B) was considered as a means for
increasing the chance of intercalation and exfoliation of clay plates into PTA chains during in
situ polymerization process. For this purpose, the hydroxyl groups were converted to
corresponding benzenesulfonate groups via the reaction with benzenesulfonyl chloride.
Subsequent nucleophilic substitution reaction of this activated intermediate with sodium azide
produced the desire clickable azidated clay. The FTIR spectra of Cloisite 30B before and after
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modification reaction were depicted in Figure 4. The appearance of a new absorption band at
2120 cm-1 clearly confirmed the presence of azide groups on MMT-N3.
Figure 4. FTIR spectra of (A) Cloisite 30B and (B) MMT-N3
PTAs were prepared through reaction of ASBO with propyn-terminated monomers in a way that
a 1:1 molar ratio of azide and propyn functions was attained (Table 1). The click polymerization
reaction of alkynated and azidated soybean oils was fully investigated by Shah and co workers
[47,50]. Properties of PTA networks obtained under thermal condition were compared with those
polymers prepared in the presence of CuAAC as catalyst. Higher yield at shorter polymerization
time, more homogenous network, cleaner polymers without entrapped catalyst and narrower
range for decomposition temperature were obtained under thermal polymerization condition.
Due to aforementioned advantages and intended biomedical application of the final polymers,
thermal (catalyst free) procedure was selected in this study. To modify final properties of coating
materials, APS and MMT-N3 were added to the base starting materials. All of the prepared PTA
coatings showed complete curing with high gel content (up to 98%); therefore, the applied
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polymerization procedure was adequate for successful formation of crosslinked networks. The
representative FTIR spectra for PTA1, PTA3 and PTA5 are presented in Figure 5. Successful
formation of 1,2,3-triazole rings during polymerization reaction was confirmed by the detection
of peaks at about 3130 cm-1 (stretching vibration of C-H on the triazole ring), 1655 and 1460
cm-1 (stretching vibration of C=C and C=N in the triazole ring) and 1045 cm-1 (stretching
vibration of C-N in the triazole ring overlapped with C-N in TPUM). For PAT3 sample with
added APS the most of basic structural peaks of pyridinium salt were merged with polytriazole
related peaks, however, the presence of this salt in the coating material was clearly indicated by
the detection of the small peak appeared at 2030 cm-1 related to N3 counter anion. For clay
containing sample, PAT5, the peaks at 798 cm-1 (Si-O stretching) and 623 cm-1 (Al-O and Si-O
out of plane bending) were obvious, however, the peak related to in plane stretching vibration of
Si-O at 1047 cm-1 was merged with vibration of C-N bonds of triazole rings. In all of the
aforementioned materials the broad peaks at about 1740 cm-1 were related to hydrogen bonded,
and non-hydrogen bonded ester and urethane carbonyl groups. Also the broad peaks at about
3420 cm-1 were attributed to the stretching vibration of hydrogen bonded and non-hydrogen
bonded urethane N-H derived from TPUM and hydroxyl groups of ASBO.
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Figure 5. FTIR spectra of (A) PTA1, (B) PTA3, and (C) PTA5
The chemical routes followed for the preparation of necessary starting materials are collected in
Scheme 1.
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Scheme 1: Synthetic routes for the preparation of starting materials
EDX, XRD and TEM studies on PTA6 nanocomposite
To find a preliminary insight regarding proper distribution of APS and MMT-N3 throughout the
coating materials, EDX analysis was run on the cross-section of PTA6. The very good overall
dispersion of APS and MMt-N3 in polymer matrix was confirmed by visualization of bromine
and silicone atoms as shown in Figure 6.
Figure 6. EDX analysis data for PTA6
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The PTA6 was also subjected to XRD analysis. The X-ray diffractograms for MMT-N3 and its
nanocomposite (PTA6) are shown in Figure 7. A peak with 2
N3 corresponding to d-spacing of 1.84 nm. After in situ polymerization in the presence of MMT-
N3 a small peak at 2 value of 3.7º was recorded. This peak corresponds to intergallery spacing
θ value of 4.8º recorded for MMT-
θ
of 2.39 nm. A 30% increase in gallery spacing was indicated the intercalation of clay with PTA
chains. In fact, the whole nanocomposite consists of both exfoliated and intercalated clay plates.
The incomplete exfoliation of clay may be related to partial contribution of azide groups located
in clay plates during in situ click polymerization reaction. This finding was more elucidated by
examination of TEM micrograph of PTA6 cross-section since this method provides the most
direct measure for dispersion of nanometer scale silicate plates in PTA6 matrix (Figure 7). The
dark lines represent clay layers and the bright areas indicate PTA6 matrix. Investigation of this
figure shows that in addition to discrete clay plates, there are clay layers preserved their stacking
structure but with higher gallery spacing. The former indicates exfoliation and the later confirm
the intercalation of clay with polymer chains.
Figure 7. (A) X-ray diffractograms of MMT-N3 and PTA6, (B) TEM of PTA6
Thermo-mechanical property and thermal stability of PTAs
Thermal transitions and relaxations related to the structure of prepared PTAs with and without
APS and MMt-N3 modifiers were analyzed by DMA. The experiments were performed in torsion
mode. The variation in storage modulus (G
′), loss modulus (G
″) and loss tangent (tan δ) as a
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function of temperature was monitored (Figure 8). The thermal transition temperatures and
calculated cross-link density of poly1,2,3-triazole networks are also collected in Table 2.
According to Figure 8, and noting the variation of G′ vs. temperature it can be concluded that the
neat polymers (PTA1 and PTA2) and APS containing polymers (PTA3, PTA4) had single phase
structure since just one glass transition in the range of 58 to 80 °C was detected for them. This
observation confirms the phase mixing of soft segment, originated from fatty ester structure of
soybean oil, and hard segment, consisting of 1,2,3-triazole rings and urethane linkages.
According to Table 2, Tg value of PTAs was increased with increasing the functionality of
propyn containing monomer. This was attributed to higher crosslink density of PTA1 than PTA2
(Table 2). As well, the breath and height of the tan
δ curve can be used for qualitative
interpretation of crosslink density and flexibility of polymeric coatings. It is anticipated that the
flexibility decreases and crosslink density increases with increasing functionality of propyn
containing monomer. Monitoring of tan
δ curves confirmed this expectation. The tan δ peak not
only shifted to higher temperature but also broadened for PTA1 in comparison to PTA2.
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Figure 8. (A) Storage modulus, (B) loss modulus and (C) tan δ vs temperature curves for PTAs
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Table 2. DMA data of PTAs
Sample
code
G
′
at glassy
state
Tg
(°C)
tan
at Tg
δ
G
′
at the onset of
Temperate atthe
onset of rubbery
region (K)
νc
rubbery region
(mol/m3)
× 10-5
(MPa)×10-3
(MPa)×10-3
PTA1
PTA2
PTA3
PTA4
PTA5
PTA6
4.90
4.47
3.59
3.75
4.68
4.09
71.8 0.054
57.8 0.050
79.7 0.041
60.2 0.079
113.4 0.057
142.5 0.037
3.38
2.77
2.49
2.41
2.78
2.44
361.5
347.9
369.7
384.4
427.9
200.7
11.20
9.57
8.10
7.50
7.81
6.20
With introduction of APS in the formulation of PTAs, the G' decreased, and Tg increased. The
reduction in G' value might be related to the decrease in hard domain formation and
consequently decrease in chemical crosslink density due to the presence of monofunctional azide
molecule. Meanwhile, with introduction of APS, the quaternary pyridinium groups with high
ability for Columbic type attraction created in the system. The restriction of chain mobility as the
result of physical crosslinking led to an increase in Tg for PTA3 and PTA4 in comparison to
PTA1 and PTA2 [51].
For clay containing nanocomposites, PTA5 and PTA6 samples, introduction of clay resulted in
an increase in Tg. Higher Tg of these samples was due to restriction of polymer chains mobility
as the result of hydrogen bonding type interaction of triazole and urethane groups of PTA chains
with silanol moieties of clay plates. Considerable increase in Tg value of these samples,
especially for PTA6 that contain both APS and MMT-N3 moieties was not only indicated good
dispersion of clay moieties in PTA matrix, but also showed substantial ability of ionic center of
AP to interact with each other. However, crosslink density was decreased for both of these
samples that again was attributed to the presence of monofunctional APS and restriction of clay
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plates for effective collusion of azide and propyn functions during in situ polymerization
reaction.
Thermograms (TGA and DTGA curves) for different formulations of PTAs are collected in
Figure 9. A multistep thermal degradation pattern was detected for the prepared polymers. The
initial weight loss of these samples (at about 300 ºC) was related to the degradation of thermally
labile urethane linkages. Since the aliphatic urethane bond is more stable than aromatic
analogous, the initial thermal decomposition of samples made from BPUM (PTA2, PTA4) was
started at higher temperature than those made from TPUM (PTA1, PTA3, PTA5, PTA6). The
weight losses at higher temperatures up to 500 ºC were attributed to the degradation of aliphatic
hydrocarbon structure of soybean oil, aliphatic or aromatic structure of propyn containing
monomers and 1,2,3-triazole rings. In this region, the sample containing benzene rings and more
concentration of 1,2,3-triazole rings (PTA1, PTA3, PTA5, PTA6), exhibited higher degradation
temperature. Meanwhile, the slope of degradation curve was gentler for these samples in
comparison to PTA2 and PTA4 samples. Introduction of APS into the formulation of coating
materials caused a mild decrease in thermal stability up to 500 ºC. This phenomenon was
attributed to the combination of dealkylation and Hoffman type elimination of quaternary
pyridinium salt moiety of APS [52,53]. The char yield at 600 ºC was increased for those samples
made from TPUM, which contain more concentration of 1,2,3-triazole rings. The coatings
containing MMT-N3 were also exhibited better thermal stability than neat polymers due to the
formation of clay char that acts as a mass transfer barrier and insulator between the bulk and
surface of polymer. The PTA6 sample containing both APS and MMT-N3 and composed of
aromatic tri-functional propyn groups which led to highest concentration of 1,2,3-triazole rings
exhibited the best thermal stability.
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Figure 9. (A) TGA, and (B) DTGA curves of PTAs
Considering the thermal curing procedure, thermal stability and thermo-mechanical properties of
these polymers, it is possible to select the range of substrates may be coated by PTAs. Non-
flexible medical devices which can withstand high temperature up to 100 ºC are within the
suitable substrates. Examples may include but not limited to polymers such as polycarbonate,
poly(methyl methacrylate), polypropylene, poly(ethylene terephthalate), polyurethane,
polytetraflouroethylene, polyvinylidene fluoride, metals such as stainless steel, titanium alloy,
Cu-Ni alloy, aluminium and glass.
Physical properties of PTAs
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For having proper biocidal activity, the microorganism should spread and come into contact with
biocidal moieties chemically anchored on the coating surface. Three primary factors that
influence bacterial adhesion on a polymeric surface are the nature of the environment, type of
microorganism and the properties of the material. Each one of these factors is in turn affected by
several other parameters [54]. Among material’s related issues, surface hydrophilicity and
surface charge play important roles. Hydrophilic surfaces are more suitable for microorganisms,
which are hydrophilic under physiological condition. Su and coworkers reported higher surface
hydrophilicity and higher negative charge density for gram negative bacteria such as
Pseudomonas aeruginosa than gram positive bacteria such as Staphylococcus aureus [55].
Therefore, gram negative bacteria are more prone to contact with hydrophilic surface bearing
positive charge. The surface and bulk hydrophilicity of prepared polyurethanes were studied
through measurement of their static water contact angle and the amount of equilibrium absorbed
water. Data are collected in Table 3.
PTA1 sample made from aromatic TPUM monomer showed lower surface hydrophilicity (higher
contact angle) than PTA2 made from aliphatic BPUM monomer due to the presence of nonpolar
aromatic rings in this polymer. The introduction of hydrophilic APS with quaternary pyridinium
salt moiety into the formulation of coatings enhanced their surface hydrophilicity considerably.
The same was happened by introduction of MMT-N3 into the formulations. This was due to the
presence of build-in quaternary ammonium salt in modified clay gallery and high concentration
of silanol groups with ability of interacting with water molecules through hydrogen bonding.
The variation of EWA was also according to the general trend detected for contact angle of
coating materials. It is expected that samples with higher surface and bulk hydrophilicity show
higher biological activities since there is higher chance for surface exposure of polar active
functions chemically anchored to PTAs backbone.
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Table 3. Bulk and surface hydrophilicity, hardness and adhesion strength data of PTAsa
Sample contact angle
EWA
(%)
Pendulum hardness Adhesion strength
code
(º)
(s-1)
(MPa)
PTA1
PTA2
PTA3
PTA4
PTA5
PTA6
81.8 ± 2a
72.8 ± 6b
56.8 ± 3c,d
52.8 ± 2d
61.6 ± 3c
55.0 ± 3cd
1.0 ± 0.2a
1.4 ± 0.3a
16.7 ± 1.1b
24.82 ±0. 9c
2.8 ± 0.3a
14.3 ± 1.1b
256 ± 6a
169 ± 5b
228 ± 2c
191 ± 8d
270 ± 5a
169 ± 6b
1.65 ± 0.07a
1.15 ± 0.07b
0.85 ± 0.07c
0.65 ± 0.07c
1.35 ± 0.07b
0.75 ± 0.07c
aAccording to analysis of variances the difference between quantities with similar superscripts (a,
b, c and d) is not significant (p > 0.05) for data of each column.
The importance of adhesion (the ability of a coating to resist removal from the surface to which
it is applied) and significant of hardness (the ability of a coating to resist permanent indentation,
scratching, cutting, and penetration by a hard object) are self evident. There are numerous
methods for determination of these important factors. Measuring hardness with a pendulum
damping device and quantifying the adhesion of coating by determination of pull-off strength
were selected in this research. Considering the chemical nature of the PTAs, materials with
negatively charged surface or rich with polar functional groups are more likely to be covered
with these polymers. The former provides Columbic type attraction with embedded positive
charge of prydinium group of APS, and the latter offers hydrogen bonding type attraction with
1,2,3-triazole rings of PTAs. The aforementioned functions may present on the substrates surface
or can be produced through different surface treatment procedures. In the present work, the
substrate made from surface treated Aluminium was selected to test hardness and adhesion
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strength of PTA coatings. Results are collected in Table 3. PTA1 with aromatic benzene rings
and higher crosslink density (higher concentration of 1,2,3-triazole rings) than PTA2 showed
higher hardness. Marked improvement in mechanical properties such as hardness was reported
for clay containing nanocomposites due to reinforcing effect of dispersed clay plates. In the
present work, introduction of MMT-N3 was not changed the hardness of coating significantly
(PTA1 vs. PTA5). This behaviour was in accordance to DMA data and it could be attributed to
the reduction of crosslink density for PTA5 upon introduction of MMT-N3. The hardness was
also reduced slightly for samples containing APS in comparison to those corresponding samples
without added salt. Again, reduction of crosslink density was main determining factor.
Almost the same trend was detected for the measured adhesion strength of PTA coatings with
different formulations. Generally, it is expected that incorporation of organically modified clay
into the polymer matrix results in an improvement of the adhesion strength due to the increased
cohesive strength of the base polymer [56]. As well, adhesion promoting property of polymeric
and low molecular weight quaternary alkyl ammonium compounds as a result of increased
Coulombic attraction of coating material and substrate is well documented [48].The reduction in
adhesion strength of PTA coatings upon introduction of MMT-N3 and APS was mainly devoted
to the reduction of crosslink density, which was correspond to a decrease in number of 1,2,3-
triazole rings throughout the polymer matrix. It is worth to mention that the adhesion strength of
the prepared materials even with APS and MMT-N3 additives is much higher than similar
coatings made from vegetable oil building blocks [48,57]. The very good adhesion strength of
PTA coatings can be attributed to strong hydrogen bonding type interaction of triazole rings
with the Al–OH groups on the Aluminium surface [58].
Biological activity of PTAs
If an infection is to be inhibited at all, the highest chance of doing so will be in the initial phase
where the bacterium is in its most vulnerable state. Functionalization of materials surface with
tethered biocidals is of high interest in the health care system, as the surfaces that bear biocidal
properties are prone to prevent microorganisms' colonization. The presence of positive charge on
a surface has a negative effect on cell survival in general, since it can disturb the membrane
integrity, decrease the metabolic activity or activate the intracellular signal transduction
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