Materials Science and Engineering C 33 (2013) 153–164

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Synthesis and characterization of antibacterial polyurethane coatings from quaternary ammonium salts functionalized soybean oil based polyols Hadi Bakhshi a, Hamid Yeganeh a,⁎, Shahram Mehdipour-Ataei a, Mohammad Ali Shokrgozar b, Abbas Yari a, Seyyed Nasirodin Saeedi-Eslami b a b

Polyurethane Department, Iran Polymer and Petrochemical Institute, P.O. Box: 14965-115, Tehran, Iran National Cell Bank of Iran, Pasteur Institute of Iran, P.O. Box: 13185-1667, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 5 May 2012 Received in revised form 18 July 2012 Accepted 11 August 2012 Available online 19 August 2012 Keywords: Soybean oil Polyurethanes Quaternary ammonium salt Antibacterial Biocompatibility

a b s t r a c t In this study, a simple and versatile synthetic approach was developed to prepare bactericidal polyurethane coatings. For this purpose, introduction of both quaternary ammonium salts (QASs), with well-known antibacterial activity, and reactive hydroxyl groups on to the backbone of soybean oil was considered. Epoxidized soybean oil was reacted with diethylamine and the intermediate tertiary amine containing polyol was reacted with two different alkylating agents, methyl iodide and benzyl chloride, to produce MQAP and BQAP, respectively. These functional polyols were reacted with different diisocyanate monomers to prepare polyurethane coatings. Depending on the structure of monomers used for the preparation of polyurethane coatings, initial modulus, tensile strength and elongation at break of samples were in the ranges of 122–339 MPa, 4.6–12.4 MPa and 8.4–46%, respectively. Polyurethane coatings based on isophorone diisocyanate showed proper mechanical properties and adhesion strength (0.41 MPa) for coating application. Study of fibroblast cells interaction with prepared polyurethanes showed promising cells viability in the range of 78-108%. Meanwhile, MQAP based samples with higher concentration of QASs showed better adhesion strength, surface hydrophilicity and antibacterial activity (about 95% bacterial reduction). Therefore, these materials can find applications as bactericidal coating for biomedical devices and implants. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Over the past decades, the global concerns about the risk of bacterial infection have been growing [1]. In this regard, coatings with antibacterial activity offer an approach to limit the spread of bacterial infections in many areas such as medical devices, healthcare products, hospitals and dental office equipments, food and drug manufacturing instruments [2,3]. Various methods have been developed for the introduction of biocidal function in polymeric coating materials [2,4,5]. Impregnation of biocidal additives into the coating materials is the most frequent technique offering antibacterial activity for coatings. The common disinfectants or antibacterial additives used in this method are heavy metals (e.g., silver, copper, zinc and tin) [6–10], phenols [11], halogens (e.g., iodine) [12,13], nitric oxide [14], phosphonium and quaternary ammonium compounds [15,16], and antibiotics [17–19]. However, this approach suffers from limited service time, since the active agents are released gradually into the surrounding environment. As well, release of the low molecular weight antibacterial agents to the environments can result in human toxicity and bacteria resistance effects [20]. Preparation of polymeric coatings with chemically anchored biocides with the contact killing ability of harmful microorganisms is a growing ⁎ Corresponding author. Tel.: +98 21 48662447; fax: +98 21 44580021. E-mail address: [email protected] (H. Yeganeh). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.023

field in the area of hygiene surfaces [2–4]. This approach provides several advantages, including avoiding the release of biocides into the environment, prolonged service time of coating and increased antibacterial efficiency/selectivity arising from high local concentration of biocides at the surface of materials [1–4]. Bonding of biocidal functional moieties to the preformed polymers [20–22] or synthesis of biocidal functional monomers and their subsequent polymerization [23–26] are two main approaches considered for the preparation of such active polymeric coatings. The second approach was employed in the present work. For this purpose, quaternary ammonium salts (QAS) with well-known extensive bactericidal activity [3,21,22] were introduced into the backbone of a series of polyols, which are suitable for the preparation of polyurethane based coatings. It is worth mentioning that due to their excellent performance as coating materials for various surfaces with diverse final biomedical applications [27,28], the polyurethanebased framework has been selected in this study. In this regard, several approaches were followed to prepare antibacterial polyurethane coatings [23,29–33]. Nowadays, polyurethanes are mainly prepared from petroleumbased raw materials; however, many scientific and industrial attempts have been started to replace them with those based on renewable resources. The main achievement in this regard is devoted to the preparation of polyols from vegetable oils as widely available, inexpensive,

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biocompatible and biodegradable feed-stock [34]. Besides castor oil with inherent hydroxyl groups, soybean oil is another important vegetable oil, that after introduction of hydroxyl groups, was widely applied for the preparation of polyurethanes [35–37]. There are some interesting reports regarding the preparation of vegetable oil-based biocidal polyurethanes [6], poly(urethane-acrylate)s [8], alkyd resins [7], and polyesteramides/poly(esteramide-urethane)s [38–40] via incorporation of silver, zinc, cadmium and boron particles. Recently, Larock and coworkers studied the mechanical and thermal properties of cationic polyurethane dispersions based on castor oil, methoxylated epoxidized soybean oil and acrylated epoxidized soybean oil as the soft segments. In this work the QAS was introduced into the polyurethane backbone through acidification of tertiary amine groups of the hard segments (N-methyl diethanol amine) with acetic acid [41,42]. Preparation and application of QAS containing vegetable oils for skin and hair products [43], clay modification [44,45] and drug delivery systems [46–49] were also considered by researchers. However, a comprehensive survey of literature shows that there is no report regarding preparation and evaluation of antibacterial activity of polyurethanes from vegetable oils containing both QAS and hydroxyl groups. Therefore, chemical modification of soybean oil with dual functionality of hydroxyl groups and QAS, and subsequent polymerization leading to biocidal polyurethane coatings is considered as the main goal of the present work. All the starting materials and final polymeric coatings were characterized by conventional spectroscopic methods. Physical, mechanical and viscoelastic properties as well as biocompatibility and antibacterial properties were studied and the feasibility of these materials as bactericidal coatings was confirmed. 2. Experimental 2.1. Materials Epoxidized soybean oil (ESBO, PATSTAB 901) with a number average molecular weight of ca. 1000 and epoxy content of 3.32 mmol epoxy/kg was purchased from PATCHEM (Sharjah, UAE) and used as received. Diethylamine (DEA, >99%), benzyl chloride (BzCl) and methyl iodide (MeI) were obtained from Merck and used without further purification. Diisocyanate monomers consist of hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI), and dibutyl tin dilaurate (DBTDL) as catalyst was purchased from Merck. Zinc chloride (ZnCl2) from Merck was dried according to the procedure reported in reference [50] and ground to fine powders prior to use. Magnesium sulfate (MgSO4.7H2O), sodium bicarbonate (NaHCO3) and sodium chloride (NaCl), ethyl acetate and tetrahydrofurane (THF) all from Merck were used as received. Phosphate buffered saline (PBS) was prepared via dissolving NaCl (5.85 g), KH2PO4 (0.6 g), Na2HPO4 (6.4 g) all from Merck in distilled water and the volume adjusted to 1 l. The pH was then adjusted to 7.4 by HCl or NaOH solutions (0.2 M). Mouse L-929 fibroblast cells were supplied from Pasteur Institute of Iran. Local pathogenic Gram-positive Staphylococcus aureus (S. aureus), and Gram-negative Escherichia coli (E. coli) bacteria were also received from Pasteur Institute of Iran. 2.2. Synthesis of tertiary amine-functional soybean oil-based polyol (TAP) TAP polyol was synthesized through the ring opening reaction of epoxy groups of ESBO with DEA according to the procedure reported in reference [51]. In a 250 ml round-bottomed flask equipped with a condenser, oil bath and magnetic stirrer, were placed ESBO (20 g, 66.4 mmol epoxy groups), DEA (8.64 g, 118.2 mmol) and anhydrous ZnCl2 (2.21 g, 16.2 mmol). The mixture was stirred at 80 °C for 4 h and then allowed to cool down to room temperature. The un-reacted DEA was removed using a rotary evaporator at 60 °C

under vacuum. The residue was dissolved in ethyl acetate (50 ml) and transferred to a separatory funnel and washed three times with saturated sodium bicarbonate solution (50 ml) to remove the zinc chloride catalyst. The washing procedure was repeated with distilled water (50 ml) and saturated sodium chloride solution (50 ml). Finally, the organic layer was dried over magnesium sulfate. The yellow-brown oil of TAP polyol at 85% yield was obtained after evaporation of solvent in a rotary evaporator at 60 °C under vacuum. 2.3. Synthesis of QAS-functional soybean oil-based polyols (BQAP and MQAP) BQAP and MQAP were prepared by alkylation of tertiary amine groups of TAP polyol with BzCl and MeI respectively. For the preparation of BQAP, TAP (20 g, ~ 20 mmol tertiary amine) and BzCl (5.06 g, 40 mmol) were placed in a 100 ml round-bottomed flask equipped with a condenser, oil bath and magnetic stirrer. The mixture was stirred at 80 °C for 28 h, and then the excess of BzCl was removed via evaporation in a rotary evaporator at 60 °C under vacuum. For the preparation of MQAP, the aforementioned procedure was repeated with MeI (4.6 g, 32.4 mmol) as the alkylating agent at 38 °C for 20 h. Both polyols were freed from moisture via heating in a vacuum oven at 60 °C just prior to reaction with diisocyanate monomers. 2.4. Preparation of polyurethane coatings (XPUs) Free stand films of XPUs were prepared via one-shot reaction of either BQAP or MQAP with different diisocyanate monomers. The formulations and quantity of necessary raw materials are given in Table 1. As a general procedure, the desired amount of polyol and catalyst were dissolved in THF solvent. Then diisocyanate monomer was added, and solid content was fixed at 35 wt.%. The solution was stirred vigorously at room temperature, degassed via application of mild vacuum, and subsequently cast in a Teflon coated mould. The curing reaction was continued at 70 °C for 8 h. All samples were kept at 90 °C for 1 h for post-curing stage. 2.5. Instruments 1 HNMR spectra were recorded on a 400 MHz Bruker Instruments (model Avance 400, Germany) at room temperature using CDCl3 as a solvent. FT-IR spectra were recorded on Bruker Instruments (model Aquinox 55, Germany) in the range of 4000–400 cm −1 at a resolution of 0.5 cm −1 and signal averaged over 8 scans. Elemental analysis was performed on a C-H-N elemental analyzer (Perkin Elmer Instruments, model Series II 2400, USA). Dynamic mechanical analysis (DMA) was carried out on a Triton instrument, (model Tritec 2000, England) at temperature range from −100 to 200 °C at a

Table 1 Different formulations of polyurethanes.a Sample Code

Polyol type

Diisocyanate monomer type

Polyol (g)

DBTDL (g)

Diisocyanate monomerb (g)

Gel Content (%)

XPU1 XPU2 XPU3 XPU4 XPU5 XPU6

BQAP BQAP BQAP BQAP MQAP MQAP

HDI IPDI TDI MDI IPDI TDI

5.00 5.00 5.00 5.00 5.00 5.00

1.5 × 10−2 1.5 × 10−2 1.5 × 10−2 1.5 × 10−2 1.5 × 10−2 1.5 × 10−2

1.27 1.68 1.32 1.90 1.51 1.18

88.6 89.3 92.9 94.5 91.4 96.2

a XPU: Crosslinked polyurethane, BQAP: Benzyl diethyl ammonium chloride-functional soybean oil-based polyol, MQAP: Diethyl methyl ammonium iodide-functional soybean oil-based polyol, HDI: Hexamethylene diisocyanate, IPDI: Isophorone diisocyanate, TDI: Toluene diisocyanate, MDI: methylene diphenyl diisocyanate. b [NCO]/[OH]=2.5/1.

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heating rate of 5 °C/min and frequency of 1 Hz. The dimensions of the samples were 30 × 10 mm 2. The values of storage modulus, loss modulus, and tan δ versus temperature were recorded for each sample. Mechanical properties including tensile strength, initial modulus, and elongation at break were determined from stress–strain curves with a MTS tensile tester model 10/M at a strain rate of 10 mm/min. Measurements were performed at ambient temperature with a film thickness of about 1 mm and stamped out with an ASTM D638 die. Data reported was an average of at least 5 measurements. Scanning electron microscope (SEM, Tescan, Vega II, Czech) equipped with an energy dispersive X-ray analyzer system (EDXA, Oxford Instrument, INCA, England) was used for EDX analysis. Hardness of prepared samples was determined by a pendulum hardness tester (Elcometer, model 3034, England) on persoz mode according to ASTM D4366 test method. Adhesion of polyurethanes on the treated aluminum substrate was also studied by a pull off adhesion tester (Erichsen testing equipments, model 525, USA) according to ASTM D4541 test method. 2.6. Measurements The hydroxyl number and acid number of prepared polyols were determined according to ASTM D4274 and ASTM D4662 test methods. The gel content of polyurethanes was evaluated. For this purpose, the samples were dried under vacuum at room temperature for 24 h and weighed. The samples were then extracted by THF in a Soxhlet extractor for 24 h. The insoluble part was dried at 70 °C and weighed. The gel content was defined as follows: Gel content % ¼ ðW d =W i Þ  100

ð1Þ

, where Wd is the weight of dried sample after extraction and Wi is the initial weight of the sample. The crosslink density (ν) of prepared polyurethanes was calculated using DMA data and according to the following equation: ′

E ¼ Φνc RT

ð2Þ

, where E′ is storage modulus at rubbery plateau region, Φ is front factor and assumed to be unit for ideal rubber, R and T are gas constant and absolute temperature, respectively. Surface hydrophilicity of prepared polyurethanes was determined by measurement of water droplet contact angle. Six different water droplets were placed on the surface at different positions. The contact angle was determined via running ImageJ 1.44p software on the digital pictures taken from interfaces of films and droplets. The values reported are an average of six measurements. The bulk hydrophilicity of prepared samples was evaluated based on equilibrium water absorption (EWA) of samples. The completely dried and accurately weighed films were soaked in distilled water at room temperature until the equilibrium was attained (about 48 h). The weight of swollen sample was determined after blotted with filter paper to remove the surface liquid. EWA was calculated using the following equation: EWAð% Þ ¼ ½ðW s −W d Þ=W d   100

ð3Þ

, where Wd and Ws are the weights of dry and swollen samples, respectively. 2.7. Biocompatibility assays Biocompatibility of prepared polyurethanes was studied by either microscopic investigation of fibroblast cells morphology after direct contact with samples or tetrazolium dye-based colorimetric assay (MTT assay). Samples were sterilized using an autoclave at 120 °C for 15 min. For the first method, mouse L929 fibroblast cells were

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pre-cultured in 24-well plates (1 × 10 4 cells/well) using RPMI-1640 growth medium supplemented with 10% fetal bovine serum (FBS) at 37 °C and atmosphere of 5% CO2 for 24 h. The cells were then exposed to the sterilized samples (5 × 5 mm 2), which placed in the center of each well and incubated at 37 °C for another 24 h. A well containing cells and growth medium with no sample was set up as negative control. The cell growth characteristics and morphological changes as a cytotoxicity sign were recorded using a TMS inverted optical microscope equipped with a Sony DSC-W7 camera. The test was repeated three times for each sample. The relative viability of the mouse fibroblast cells contacted with either polyurethane films (direct contact) or their leachates in growth medium (indirect contact) was determined by MTT assay. The MTT solution was prepared via dissolving MTT dye (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide, Sigma) in phosphate buffered saline (PBS) at a concentration of 0.5 mg/ml. The solution was filtered through a 0.45 μm filter in order to sterilize and remove small amount of insoluble residues and stored at 2–8 °C for frequent use. Then, pre-cultured cells were seeded into a 96-well plate with a density of 1 × 104 cells/well in RPMI-1640 medium containing 10% fetal FBS, and incubated at 37 °C and atmosphere of 5% CO2 for 24 h. The polyurethane films were extracted using RPMI-1640 medium (1 ml/cm 2 of film) at 37 °C for one week. For direct contact test, cells were exposed to the extracted films (2 × 2 mm 2) and incubated at 37 °C for another 24 h. While for indirect contact test, the culture medium of each well was replaced by 180 μl of the films extracts, and then 20 μl FBS was added to each well. Incubation at 37 °C was continued for 24 h. For each of direct and indirect methods, the culture medium was discarded and 100 μl of the MTT solution was added into the wells. The cells were incubated at 37 °C for 5 h. Then, the MTT solution was removed and the purple crystals of formazan were dissolved by the addition of 100 μl of isopropanol or DMSO (Merck) per well. The plates were incubated at 37 °C for 15 min prior to absorbance measurements. The optical density (OD) of formazan in the solution was measured at 545 nm using a multiwall micro-plate reader (ELISA reader, Organon Teknika, The Netherlands). A seeded well with no sample and a well containing 100 μl of isopropanol were used as negative and positive controls, respectively. Reported values are the means of five replicates. The percentage of relative cell viability was calculated according to following equation:

Cell Viability % ¼

OD Sample −OD Positive OD Negative

Control

 100

ð4Þ

Control

, where OD is mean optical density. 2.8. Antibacterial assays Antibacterial activity of polyurethanes was studied against both E. coli and S. aureus according to agar diffusion and shaking flask methods. For agar diffusion method, samples were exposed to bacteria in solid media (nutrient agar) and the zone of inhibition underneath and around each sample was monitored. For this propose, prepared polyurethanes were cut into 1 cm disk and placed on an LB agar plate (Himedia) seeded with 1 × 10 4 CFU of E. coli for 5-10 min. After incubation at 37 °C for 24 h, inhibition zone for bacterial growth was detected visually. In shaking flask method, the antibacterial activity of samples was evaluated quantitatively by measuring OD600nm [52,53]. Bacteria were pre-cultured overnight at 37 °C to prepare a culture in logarithmic growth phase for the antibacterial test. Then, dick samples was dipped into a flask containing 10 ml of LB broth (peptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l, pH 7.4) and 1.5 × 104 CFU of E. coli or S. aureus. The flask was incubated in a rotary shaker at 200 rpm overnight at 37 °C.

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After that, the OD of culture solution (200 μl) was measured at 600 nm using a biophotometer (single beam, Eppendorf, Germany). A flask containing bacteria and no sample was used as negative control. Another flask with sample and no bacteria was employed as positive control. Reported values are the means of three replicates. The percentage of bacterial reduction was calculated according to the following equation:

Bacterial Reduction % ¼

OD Sample −OD Positive OD Negative

Control

 100

ð5Þ

Control

2.9. Statistical analyses Statistical analyses were performed via PASW Statistics program package, version 18 (SPSS Inc., Chicago, IL, USA). Comparison of obtained data for different samples was performed with One-Way ANOVA with Tukey or Bonferroni posthoc tests. The significance level was set at p > 0.05. 3. Results and discussions 3.1. Synthesis and characterization of functional polyols The reaction of ESBO with DEA was utilized for the simultaneous introduction of tertiary amine and hydroxyl groups into the soybean oil backbone. Different catalysts under various conditions have been used for the ring-opening reaction of epoxy groups of ESBO with amines [51,54,55]. Due to ease of reaction and acceptable conversion of epoxy groups, anhydrous zinc chloride was selected as catalyst in this study [51]. Subsequent alkylation of intermediate tertiary amine containing polyol, TAP, with either methyl iodide or benzyl chloride led to soybean oil based polyols (BQAP, MQAP) with well-known antibacterial QAS (Scheme 1). The chemical identity of TAP polyol was evaluated by 1H NMR and FTIR spectroscopies as well as elemental analysis. Fig. 1a shows 1H NMR spectrum and assigned chemical structure of TAP polyol. The amounts of unreacted epoxy groups of the oil was determined via comparing the integration of peaks at 2.7–3.1 ppm and 5.1 ppm corresponding to the protons of epoxy groups and methene (–CH–) proton of the glycerol moieties of the oil backbone, respectively. The yield of ring opening reaction was calculated based on the reduction of epoxy groups' content. 1H-NMR analysis showed a conversion of 24.8%, while elemental analysis resulted in 17.6% conversion. The results were in accordance to Biswas report [51]. Fig. 2a shows FTIR spectrum of TAP polyol. The peak at about 3262 cm −1 was assigned to the stretching vibration of O–H bond produced via aminolysis of ESBO epoxy groups. The strong peaks at 1743, 1161 and 1107 cm −1 were due to stretching vibration of C_O and C\O bonds of fatty ester structure. Stretching vibrations of aliphatic C\H groups were observed at 2925 and 2854 cm −1. The corresponding bending vibrations of C\H groups were detected at 1463 and 1378 cm −1. Meanwhile, the peak at 1575 cm −1 was due to stretching vibration of C\N bonds. The products of alkylation reaction of TAP polyol were also identified by 1H-NMR and FTIR spectroscopies. 1H NMR spectrum of BQAP is given in Fig. 1b. According to Fig. 1b, the aromatic and benzylic C\H groups appeared at 7.2–7.4 and 4.4 ppm, respectively. As well, the peak of methene group attached to amine nitrogen atom at 2.6 ppm disappeared and was shifted to weaker filed (3.7 ppm). This phenomenon can be related to deshielding effect of quaternized amine groups. Due to the same reason, the peak of methylene groups at α position of ammonium nitrogen atom was shifted from 2.4 to 3.4 ppm. Besides, the peak of methyl groups at β position of ammonium nitrogen atom (1.0 ppm) disappeared after quaternization reaction, and shifted to weaker field and merged with the peak of

Scheme 1. Synthesis route for preparation of functional polyols from ESBO.

methylene groups of oil backbone at 1.3 ppm. The yield of alkylation reaction was calculated as 23.3% by comparing the peak area of benzylic CH2 group with the methene group of glycerol moieties of the oil backbone. FTIR spectrum of BQAP is shown in Fig. 2b. Small peaks at 3050 and 1627 cm −1 were related to stretching vibration of aromatic C\H and C_C bounds of benzylic moieties. Meanwhile, the corresponding peak of stretching vibration of C\N bond at 1575 cm −1 disappeared after quaternization of tertiary amine groups. It is widely known that upon quaternization of alkyl amines, the C\N bond order increases and the corresponding FTIR peak shifts to higher frequency [56,57]. For BQAP, the C\N bond stretching vibration peak at 1575 cm −1 disappeared, because it shifted to higher frequency and appeared as a small shoulder at about 1630 cm −1. 1 H-NMR spectrum of MQAP is given in Fig. 1c. The peaks of methene groups attached to ammonium amine nitrogen atom appeared at 3.7 ppm. The peak of methyl group at the α position of ammonium nitrogen atom arising from methyl iodide moiety appeared at 3.4 ppm and was merged with the peak of methylene groups at the same position of ammonium nitrogen atom. The yield of alkylation reaction was calculated as 79.3%, by comparing the peak area of methene group attached to ammonium nitrogen atom with methene group of glycerol moiety of the oil backbone. Fig. 2c

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157

Fig. 1. 1H NMR spectrum of TAP (a), BQAP (b) and MQAP (c) in CDCl3.

shows FTIR spectrum of MQAP. Similar to BQAP, the peak related to the stretching vibration of C\N bond at 1575 cm −1 disappeared after quaternization of tertiary amine groups. To use these polyols for the preparation of polyurethanes, the OH number of TAP, BQAP and MQAP was determined as 81, 68 and 63 mg

KOH/g, respectively. Consequently, the equivalent weight of these polyols was calculated as 693, 825 and 890, respectively. The increase in equivalent weight of quaternized polyols with respect to TAP polyol can be attributed to the increase of molecular weight after quaternization reaction. Meanwhile, due to higher yield of alkylating

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Fig. 2. FTIR spectra of TAP (a), BQAP (b) and MQAP (c) and XPU2 sample (d).

reaction for MQAP than BQAP, higher equivalent weight was calculated for MQAP. Coatings with appropriate flexibility, good adhesion to different substrates and excellent weathering resistance can be obtained by using a suitable selection of aromatic or aliphatic diisocyanates. Because of the strong tendency of rigid aromatic moieties to pack efficiently and the presence of hydrogen bonding between urethanederived groups, urethane segments tend to self-organize to form hard domains within the polymer macromolecular assembly [27]. Each type of diisocyanate has a different intrinsic ability to form such microphase structures. Aromatic diisocyanates, present a fairly rigid molecular structure due to delocalization of the π electrons throughout the aromatic rings, therefore impeding rotation of the C–C bonds leading to more efficient molecular packing and consequently higher level of mechanical properties. However, in terms of the durability of the coatings, aromatic systems are disadvantageous, since they have a stronger tendency to yellow and degrade photochemically [27]. Thus, aliphatic systems are employed in highly durable applications. Meanwhile, it has been observed that aromatic diisocyanates led to potentially carcinogenic degradation products. Therefore aliphatic diisocyanates for the synthesis of biomedicalgrade polyurethanes is considered as a solution for this issue [28]. MQAP and BQAP were reacted with different diisocyanate monomers under proper condition to obtain final polyurethane coatings with widespread properties.

Fig. 3. Storage modulus (a), loss modulus (b) and tan δ (c) vs. temperature for different polyurethanes.

The hydroxyl value of the prepared polyols was not so high, due to low overall conversion of epoxy groups of ESBO, therefore, excess amount of diisocyanate monomers was utilized ([NCO]/[OH]= 2.5/1) in all formulations in order to improve mechanical properties as a result of extra crosslinking through allophanate bonds. Different formulations of polyurethane networks (XPU1-6) were collected in Table 1. The structure of the XPUs was confirmed by FTIR spectroscopy. The representative FTIR spectrum of XPU2 is shown in Fig. 2d. The stretching vibrations of different types of C = O groups showed respective peaks at 1737, 1723, 1690 and 1640 cm−1 .The stretching vibration of urethane N–H groups were detected as a weak peak centered at Table 2 DMA data of polyurethanes. Sample Code

Tgs (°C)

Tgh (°C)

Tan δ at Tgs

E′ at rubbery region (MPa)

νc at rubbery region (MPa)

XPU1 XPU2 XPU3 XPU4 XPU5 XPU6

−23 13 26 11 8 17

– 87 161 146 83 148

0.18 0.16 0.11 0.36 0.19 0.15

3.4 1.1 1.2 0.5 8.4 8.3

1092 331 292 126 2413 2078

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Table 4 Hydrophilicity, hardness and adhesion strength of polyurethanes.1 Sample code

Contact angle (θ)

Water absorption (%)

Pendulum hardness (s−1)

Adhesion strength (MPa)

XPU1 XPU2 XPU3 XPU4 XPU5 XPU6

67 ± 1a 81 ± 2b 75 ± 1c 83 ± 2b 75 ± 3a 62 ± 2a

2.7 ± 0.8a 2.3 ± 1.2a,b 0.4 ± 0.4b 0.3 ± 0.2b 3.6 ± 0.9a 0.5 ± 0.4b

15 ± 0a 36 ± 6b 77 ± 7c 62 ± 3c 35 ± 4b 70 ± 7c

0.32 ± 0.04a,b 0.36 ± 0.03b,c 0.26 ± 0.01d,a 0.24 ± 0.02d 0.41 ± 0.02c 0.32 ± 0.03a,b

1 According to analysis of variances (p ≤ 0.05) the difference between quantities with similar superscripts (a, b, c and d) is not significant for data of each column.

Fig. 4. Stress-strain curves for different polyurethanes.

3340 cm−1. The absence of isocyanate and hydroxyl groups peaks at 2270 and 3430 cm−1 in the FTIR spectrum of final polymers and high gel content of cured samples (Table 1) confirmed complete reaction of active groups in the final coating materials.

3.2. Viscoelastic and mechanical properties Thermal transitions and relaxations related to the structure and morphology of prepared polyurethanes were analyzed using DMA. The experiments were performed in tensile mode as a function of temperature from − 100 to 200 °C. Variation of the storage modulus (E′), loss modulus (E″) and loss tangent (tan δ) as a function of temperature is shown in Fig. 3. The thermal transition temperatures of polymers can be seen in Table 2. According to Fig. 3, all samples except that prepared from HDI (XPU1) showed two-phase structure as two thermal transitions were observed for them. The first transition was related to the soft segment originated from fatty ester structure of soybean oil with glass transition temperature (Tgs) in the range of − 23 to 26 °C. The second transition in the range of 83 to 161 °C was associated to the glass transition of hard segments (Tgh) consisting of urethane and allophanate groups of polyurethanes. As expected, samples based on aromatic diisocyanate monomers showed higher Tgs and Tgh than those based on aliphatic diisocyanate monomers. This phenomenon was related to the dipole–dipole interaction of aromatic groups of polymers [27]. No glass transition of hard segment was observed for XPU1 sample made from HDI due to higher possibility of phase mixing of two soft and hard segments with similar chemical structures. Also, due to the higher ionic interactions and therefore stronger physical crosslinking in polyurethanes based on MQAP with higher concentration of QAS, higher storage modulus at glassy state was detected for XPU5 and XPU6 samples in comparison to XPU2 and XPU3 ones [58]. The crosslink density (νc) of all samples was also evaluated based on DMA data. Generally, the samples made from MQAP showed higher νc values than those prepared from BQAP. This observation can be attributed to lower reactivity of hydroxyl groups of BQAP due to higher steric hindrance of bulkier benzyl Table 3 Tensile properties of polyurethanes.1 Sample code

Initial modulus (MPa)

Stress at break (MPa)

Elongation at break (%)

XPU1 XPU2 XPU3 XPU4 XPU5 XPU6

122.5 ± 10.2a 155.7 ± 11.2b 184.7 ± 10.1c 338.7 ± 10.9d 167.4 ± 7.3b,c 194.3 ± 12.8c

4.6 ± 0.4a 6.8 ± 0.6b 7.8 ± 0.4b 12.4 ± 1.0c 7.7 ± 0.5b 8.2 ± 0.5b

24.5 ± 3.2a 39.8 ± 3.9b 24.7 ± 2.7a 8.4 ± 0.9c 41.8 ± 2.1b 23.4 ± 3.1a

1 According to analysis of variances (p≤ 0.05) the difference between quantities with similar superscripts (a, b, c and d) is not significant for data of each column.

moieties of quaternary ammonium groups. For BQAP based polyurethanes, there was no considerable difference between νc values of XPU2-4 samples; however, much higher νc value was calculated for XPU1 sample. This observation may be a result of more compact single phase structure of system arose from higher compatibility of components. The mechanical properties of all polyurethanes were determined via measuring initial modulus, tensile strength and elongation at break at ambient temperature. The results are collected in Fig. 4 and Table 3. The samples showed widespread mechanical properties depending on the structure of polyols and diisocyanate monomers used for their preparation. The initial modulus, tensile strength and elongation at break were in the ranges of 122-339 MPa, 4.6– 12.4 MPa and 8.4–46%, respectively. It was found that both initial modulus and tensile strength were higher for the samples based on aromatic diisocyantes than those containing aliphatic ones (XPU3-4 in comparison to XPU1-2 and XPU6 respect to XPU5) as a result of π-π interaction of aromatic rings. Meanwhile, higher elongation at break was observed for the samples based on aliphatic diisocyante monomers. As well, polyurethanes based of MQAP (XPU2 and XPU3) showed higher modulus, tensile strength and elongation at break in comparison to the polyurethanes made from similar diisocyanate but using BQAP as polyol (XPU5 and XPU6). It seems that higher concentration of ionic groups of XPU5 and XPU6 is responsible for this phenomenon.

3.3. Surface and bulk hydrophilicity The antibacterial activity of polymers with chemically anchored bactericidal functions depends on the degree of contact between bacteria and the polymer surface, since the bacteria should spread and come into contact with bactericidal moieties. It is well known that surface hydrophilicity is a determining factor for bacteria adhesion on the surface of materials [59]. Because most of bacteria such as E. coli and S. aureus are hydrophilic under physiological condition, therefore, bactericidal activity of antibacterial polymers is highly proportional to their surface hydrophilicity [60–62]. 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. Results are collected in Table 4. The surface of all samples were considered as moderately hydrophilic, since their water contact angle values were in the range of 62 to 83 degree. Polyurethanes based on MQAP showed lower contact angle and therefore higher surface hydrophilicity in comparison to those prepared from BQAP. This is due to higher concentration of hydrophilic QAS of MQAP based polyurethanes. The bulk hydrophilicity of prepared polyurethanes was low, since their water absorption values were less than 4%. Also, samples based on aromatic diisocyanate monomers showed lower water absorption values than those based on aliphatic diisocyanate monomers.

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Fig. 5. Optical microscopy of L929 cells in direct contact with samples: cells without sample (a), control sample made from TAP and IPDI (b), XPU1 (c), XPU2 (d), XPU3 (e), XPU4 (f), XPU5 (g) and XPU6 (h).

3.4. Hardness and adhesion The intended application of these new polyurethanes was as surface coatings for biomedical implants and devices, therefore, hardness and adhesion strength as most routine criterions for coating application were studied. The pendulum hardness values of samples are given in Table 4. As expected, aromatic diisocyanate based polyurethanes showed higher hardness in comparison to aliphatic ones. This result is in accordance to DMA data, as XPU3 with the lowest tan δ peak at Tgs (lowest damping factor) showed the highest pendulum hardness. As well, changing the polyol identity had no significant

effect on hardness of samples made from similar diisoynates. It shows that the pendulum hardness mainly influenced by the Tgh rather than Tgs of samples. The adhesion strength of polyurethane films spread over treated aluminum plate was measured by pull off adhesion test. The results are collected in Table 4. Samples showed adhesion strength in the range of 0.24 to 0.41 MPa depending on the structure of raw materials used for their preparation. Samples based on MQAP and aliphatic diisocyanate monomers showed higher adhesion strength in comparison to those based on BQAP and aromatic diisocyanate monomers. The superior adhesion strength of polyurethanes based on MQAP

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Fig. 6. Cell viability of L929 mouse fibroblast cells in direct contact with samples (a) and their extracted leachates in growth media (b). The control sample made from TAP polyol and IPDI.

can be attributed to the higher concentration of QAS in the backbone of polymers, which lead to increased coulombic attraction of coating and substrate. This phenomenon is in agreement with some reports about adhesion promoting property of polymeric and low molecular weight quaternary alkyl ammonium groups [63–65], and improved adhesion strength of polyurethane cationomers to glass, aluminum, and PVC substrates [66,67].

3.5. Cytotoxicity Keeping in mind the biocompatibility and bioapplicability prospects of our newly developed polyurethanes, their cytotoxic effects were studied against L929 fibroblast cells. Such a study is required to gain insight into their suitability for biomedical use. Cytotoxicity are known as a big challenge for most cationic carriers, as they can disturb the membrane integrity, decrease the metabolic activity or activate the intracellular signal transduction pathways after interaction with cells [20]. In practice, evaluation of in vitro cytotoxicity showed some sort of cytotoxic effect for high concentration of QAS [68,69]. However, it is also well recognized that low concentration of charged QAS incorporated in polymers can be helpful for improving the material biocompatibility and rendered them to be safer for clinical applications [70–73]. The cytotoxic effect of prepared polyurethanes on mouse L929 fibroblast cells was studied by either microscopic investigation of cells morphology or MTT assay. Optical microscopic images of the samples cultured with fibroblast cells are given in Fig. 5. Fig. 5a represents the original morphology of the fibroblasts before exposure to samples. Morphology of cells contacted with the control sample made from TAP polyol and IPDI is shown in Fig. 5b, while Fig. 5c–h shows the cell proliferation after 24 h incubation with samples containing QAS. The evaluation of cell morphology showed cells survived and grew

with spindle shape morphology and none of the prepared polyurethanes appeared to give off any toxic or inhibitory leachates, since cells grew to confluence on the all samples. Therefore, based on qualitative cell morphology inspection, the level of biocompatibility of polyurethane samples with or without QAS are acceptable. For better judgment, the biocompatibility of samples was determined via quantitative MTT assay. MTT assay is a widely used method for evaluation of cell viability based on the dye reduction by mitochondrial dehydrogenases in living cells to a blue-colored formazan precipitate. Cytotoxic compounds can damage the mitochondria of cells and decrease the reduction of MTT to formazan [74]. The MTT assay was carried out on the mouse fibroblast cells in contact with either polyurethanes or their extracted leachates in growth medium. The fibroblast cell viability values are collected in Fig. 6. The MTT assay showed cell viability values in the range of 80–110% demonstrating noncytotoxcicity and cytocompatibility of prepared polyurethanes. As well, no significant differences (p≤ 0.05) were detected for viability values of fibroblast cells in direct contact with samples or their extracts. The cell viabilities quantified by MTT assay are in good agreement with cell morphology observation, suggesting that these polymers are biocompatible and can be used as safe biomaterials.

3.6. Antibacterial activity Antibacterial polymers based on quaternary ammonium functions are known to be efficient against a large spectrum of microorganisms such as Gram-positive and Gram-negative bacteria, algae, fungi, etc. The mechanism of the bactericidal activity of immobilized quaternary ammonium groups is not entirely clear, however, it was anticipated that the process begins with direct electrostatic adsorption of the bacteria, with a negatively charged cell wall at physiological pH, to the surface of the polymer carrying positive charges [5,21]. Then immobilized

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Fig. 7. Results of agar diffusion test for XPU2 (a), XPU5 (b) and XPU6 (c) samples.

quaternary ammonium moieties exert their bactericidal effect through diffusion into the bacterial cell wall, binding to the cytoplasmic membrane, disrupting of membrane by alkyl groups, releasing of cytoplasmic constituents (K+ ions) and finally cause cell death [3,21,75]. Firstly, bactericidal activity of prepared QAS containing polyurethanes against E. coli was tested using agar diffusion method (Fig. 7 and Table 5). Since all active quaternary ammonium groups are covalently attached to the polymers, no zone of inhibition around the specimens was observed. Therefore, there is no possibility for release of active groups from the polymers. Meanwhile, no bacterial colonies were grown on both specimens' surface and area of agar plate under each sample. As a result, the prepared QAS containing polyurethanes have contact killing activity. Secondly, shaking flask method was performed to quantitatively evaluate the antibacterial activity of modified polyurethanes under dynamic contact condition. In this method, the specimens were shaken in bacteria suspensions overnight and the percentage of bacteria reduction was calculated by measuring OD600 nm. The results obtained from shaking flask test are given in Table 5. For comparison the bactericidal activity of a control polyurethane sample made from TAP polyol and IPDI was also evaluated. No antibacterial activity was detected for this sample. In spite of having QAS, the polyurethanes prepared from BQAP had no considerable antibacterial activity. However, the samples based on MQAP showed significant bacterial reduction in the range of

83-95% against both E. coli and S. aureus bacteria. To find the reason behind this observation, the concentration of active quaternary ammonium groups was examined by measuring the halogen counter ion content using EDXA (Fig. 8). Although the EDXA maps showed uniform distribution of halogen atoms, the concentration of quaternary ammonium groups were much higher for the XPU5 (0.53-0.60 mol%) than XPU2 (0.20-0.23 mol%) sample. It seems that concentration of QAS is a determining factor for observation of suitable antibacterial activity of the samples. The polyurethanes made from MQAP had enough active groups for showing acceptable bactericidal activity. Also, the antibacterial activity of XPU5 and XPU6 samples was higher for S. aureus than E. coil. This phenomenon is in agreement with some recent reports [23,76–78] regarding bactericidal activity of immobilized QAS. The observed difference was attributed to the cell membrane structures of S. aureus and E. coli bacteria. The multilayered cell envelope structure and more hydrophilic nature of Gram-negative bacteria is responsible for their higher resistant against bactericidal moieties [21,79]. 4. Conclusion A simple synthetic methodology was developed for the preparation of bactericidal polyurethane coatings from soybean oil as a low cost, widely available and renewable resource based raw material.

H. Bakhshi et al. / Materials Science and Engineering C 33 (2013) 153–164 Table 5 Data of antibacterial activity for polyurethanes evaluated by agar diffusion plate and shaking flask methods. Sample code

XPU1 XPU2 XPU3 XPU4 XPU5 XPU6 Controla

Zoom of inhibition (mm)

0.0 0.0 0.0 0.0 0.0 0.0 0.0

Bactria reduction (%) E. Coli

S. aureus

4.3 0.0 0.6 0.0 88.7 83.1 0.0

6.5 3.2 0.9 0.0 94.6 95.4 0.0

The control sample made from TAP polyol and IPDI.

The applied methodology enabled us to successfully functionalize soybean oil with quaternary ammonium salts and hydroxyl groups. It was observed that with proper use of alkylating agent and diisocyanate monomers, the biological and physicochemical properties could be tailored properly. Using methyl iodide as alkylating agent of intermediate TAP polyol and IPDI as diisocyanate monomer led to the most suitable polyurethane for intended application as biocompatible and bactericidal surface coating material. Due to nonleaching behavior of chemically attached bactericidal groups, high biocompatibility of final coatings, suitable mechanical properties and acceptable antibacterial activity, these materials can be considered as potential candidate for biomedical applications. Improving the overall conversion of epoxy groups of ESBO under more efficient catalytic systems, leading to polyols with higher hydroxyl values and QASs concentration is under investigation in our lab.

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