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Crosslinking of agar by diisocyanates
Accepted Manuscript Title: Crosslinking of agar by diisocyanates Authors: Amit Kumar Sonker, Mezigebu Belay, Kalpana Rathore, Kousar Jahan, Sankalp Verma, Gurunath Ramanathan, Vivek Verma PII: DOI: Reference:
S0144-8617(18)31057-9 https://doi.org/10.1016/j.carbpol.2018.08.138 CARP 14035
To appear in: Received date: Revised date: Accepted date:
7-4-2018 27-8-2018 30-8-2018
Please cite this article as: Sonker AK, Belay M, Rathore K, Jahan K, Verma S, Ramanathan G, Verma V, Crosslinking of agar by diisocyanates, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.08.138 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Crosslinking of agar by diisocyanates
Amit Kumar Sonker1, Mezigebu Belay1, Kalpana Rathore1, Kousar Jahan1, Sankalp Verma1, Gurunath Ramanathan2, Vivek Verma1, 3 1
Department of Materials Science and Engineering, Indian Institute of Technology Kanpur,
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3
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Kanpur - 208016 Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur - 208016
Centre of Environmental Science and Engineering, Indian Institute of Technology Kanpur,
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Kanpur - 208016
Corresponding author’s email: [email protected]
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Highlights
Agar based bioplastic is prepared by chemical crosslinking with diisocyanates
Efficacy of DDI (4, 4 diphenyl diisocyanate) and HDI (1, 6 hexamethylene
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diisocyanate) is compared on agar crosslinking
Crosslinked agar films show improved tensile strength and water resistance
Crosslinked films are non-toxic to blood and show cell proliferation
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Abstract
In the present study, crosslinking of agar using diisocyanate (DI) was demonstrated to limit the
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high water absorption property of agar. In addition, the efficacy of aromatic diisocyanate, DDI (4, 4 diphenyl diisocyanate) and aliphatic diisocyanate, HDI (1, 6 hexamethylene diisocyanate) on crosslinked agar properties was compared. The water uptake was successfully reduced by
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crosslinking and its minimum values observed for DDI and HDI crosslinked agar was 33.6% and 43.6%, respectively in comparison to agar (206%). The maximum tensile strength was observed for DDI crosslinked agar (45.3 MPa) which was higher than HDI crosslinked agar (30.6 MPa) and agar (31.7 MPa). The aromatic diisocyanates crosslinked agar showed better thermal resistance at higher temperature. It was observed that aromatic diisocyanate crosslinked agar more effectively than the aliphatic diisocyanate due to the higher reactivity. 1 | Page
The crosslinked agar samples were hemocompatible and show non-toxic nature for cell proliferation. Keywords:
bioplastic,
crosslinking,
agar,
cytocompatibility,
hemocompatibility.
Abbreviations DI – Diisocyanate
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DDI – 4, 4 Diphenyl Diisocyanate
1.
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HDI – 1, 6 Hexamethylene Diisocyanate
Introduction
Synthetic polymers such as low-density polyethylene, high-density polyethylene,
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polypropylene, polyethylene terephthalate, polystyrene are widely used packaging material.
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However, increasing demand and consumption of these ‘nondegradable’ polymers is promoting a negative impact on our ecosystem, which is necessitating thrust to develop their
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biodegradable counterparts with comparable properties (Bordes, Pollet, & Avérous, 2009; J.-
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W. Rhim, Park, & Ha, 2013; Shit & Shah, 2014; Siracusa, Rocculi, Romani, & Rosa, 2008; Tang, Kumar, Alavi, & Sandeep, 2012; Weber, Haugaard, Festersen, & Bertelsen, 2002).
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Among biodegradable polymers like polylactic acid, starch, cellulose etc., a considerable interest is developed in agar as potential packaging material (J. P. Lee, K. H. Lee, & H. K. Song, 1997; López de Lacey, López-Caballero, & Montero, 2014; Orsuwan, Shankar, Wang, Sothornvit, & Rhim, 2016; Phan The, Debeaufort, Voilley, & Luu, 2009; J.-W. Rhim & Ng,
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2007; Shankar, Reddy, & Rhim, 2015; Shankar & Rhim, 2016; Siracusa et al., 2008; Weber et al., 2002). Agar is a natural, biodegradable, water soluble and cost-effective polymer (Freile-
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Pelegrín et al., 2007; J. P. Lee et al., 1997; Skurtys et al., 2010). It is a polysaccharide extracted from agrophyte algae that belong to the phylum of Rhodophyta, which is found in seaweed
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(McHugh, 1987). Since it is extracted from seaweed, the use of agar as a packaging material neither cause deforestation, like cellulose, nor affect food supply chain, like starch. It is a mixture of gelling fraction agarose and non-gelling fraction agaropectin, which is sulfated and slightly branched (Phillips & Williams, 2000). Potentially, agar as packaging materials is reported earlier (Kanmani & Rhim, 2014; J.-P. LEE, K.-H. LEE, & H.-K. SONG, 1997; Phan The et al., 2009; J.-W. Rhim, 2011, 2012; J. W. Rhim, Wang, & Hong, 2013). However, its high water absorption, moderate mechanical strength, and thermal stability limit its application 2 | Page
as biodegradable packaging material. To overcome the above drawbacks, reinforcement and chemical cross linking of agar has been attempted earlier (Atef, Rezaei, & Behrooz, 2014; Awadhiya, Kumar, Rathore, Fatma, & Verma, 2016; Orsuwan et al., 2016; J.-W. Rhim, 2011; J. W. Rhim et al., 2013; Shankar et al., 2015; Shankar & Rhim, 2016). For example, Awadhiya et al. (Awadhiya, Kumar, Rathore, et al., 2016) showed change of tensile strength from 25.1 MPa for pure agarose to 60.1 MPa for agarose/bacterial cellulose composite. Shankar et al. (Shankar et al., 2015) reported an improvement of tensile strength from 45.7 MPa for pure agar
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to 52.1 MPa for lignin/agar composites. Rhim et al. (J.-W. Rhim, 2012) showed an increase in tensile strength of agar (31 MPa) for pure agar to 45 MPa for agar/ κ-carrageenan composites.
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Awadhiya et al. (Awadhiya, Kumar, & Verma, 2016) showed a reduction in water uptake by ~8 fold with improvement in mechanical and thermal properties by crosslinking agarose with citric acid. However, higher concentration of citric acid deteriorated the mechanical properties
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of agarose due to the hydrolyzing role of acid on agarose chains during crosslinking at high temperature. To overcome this, a process to crosslink agar is proposed that does not promote
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chain degradation during crosslinking.
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Diisocyanates do not promote chain scission and hence can also be used in higher
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concentration. Moreover, high temperature for crosslinking is not required in case of diisocyanates. It is also reasonable to expect the role of crosslinker geometry on the property
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of polymer (Sonker, Rathore, Nagarale, & Verma, 2017; Sonker, Tiwari, Nagarale, & Verma, 2016). Therefore, the aim of this work is to crosslink agar by diisocyanates to reduce its water absorption in addition to and improving the tensile strength and thermal stability. Further, the efficacy of aromatic, DDI (4, 4 diphenyl diisocyanate) and aliphatic diisocyanate, HDI (1, 6
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hexamethylene diisocyanate) on agar crosslinking is compared. For suitable comparison, swelling, mechanical and thermal properties of diisocyanates crosslinked agar are discussed in
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detail. Moreover, hemocompatibility and cytocompatibility of agar and crosslinked agar are studied for potential applications in biomedical field. Material and methods
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2.
2.1 Materials Agar (gelling temperature 34-36°C, pH 6.0-7.0, dissolution in water at 85°C) was purchased from Hi Media, India. 4, 4 diphenyl diisocyanate (DDI) and hexamethylene diisocyanate (HDI) were procured from Sigma Aldrich, India. Dimethylsulphoxide (DMSO) and tetrahydrofuran (THF) were purchased from Merck, Germany. Polypropylene (PP) petridishes were purchased 3 | Page
from Tarsons Product Pvt. Ltd. For phosphate buffered saline (PBS) solution, potassium chloride, sodium chloride, potassium dihydrogen orthophosphate, disodium hydrogen orthophosphate were purchased from Thermo Fisher Scientific (Mumbai), India. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was procured from Sisco Research Laboratories (SRL) Pvt. Ltd, India. Dulbecco’s Modified Eagle’s medium-high glucose (DMEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA) and antibiotic (penicillin and streptomycin) solution were purchased from Gibco, Thermo
2.2 Preparation of agar and diisocyanates crosslinked films
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Fisher, India. Ethanol was purchased from Merck specialties Private Limited (Mumbai).
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Agar (1 g) was dissolved in 40 mL DMSO at 80°C. The solution was then cooled down from 80°C to 10°C using an ice bath. For crosslinking, the concentrations of DDI and HDI were varied from 5% to 40% w/w (with respect to agar) with an interval of 5%. The required amount
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of crosslinker (DDI or HDI) was dissolved in 10 mL of DMSO at 10°C. The agar solution (40
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mL) was transferred into crosslinker solution (10 mL) and was mixed vigorously using a magnetic stirrer for 10-15 minutes. The solution was then poured into polypropylene petridish
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and allowed to react for 12 hours at room temperature. The petridish was kept in hot air oven
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at 60°C for 36 hours to remove DMSO. The thickness of the dried film was ~50 micron as measured using Mitutoyo micrometer. The crosslinked agar films were washed with THF to
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remove unreacted diisocyanate. Washing was performed using a mechanical shaker for 12 hours and then the films were dried at 60°C to remove THF. 2.3 Characterization of films
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Swelling study was performed to measure the amount of absorbed water according to the ISO (International Organization of Standardization) standard (ISO 62:2008, Plastics 2
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Determination of water absorption). Agar films were cut into three square pieces (2 cm
cm), weighed and immersed in deionized water for 24 hours at 25°C. The swollen samples were taken out and wiped with tissue paper to remove excess water from the film surface and
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weighed. The swelling percentage in agar and diisocyanate crosslinked agar samples is calculated by using equation (1) (1)
where WS is the weight of swollen samples and WD is the weight of dry samples. 4 | Page
Fourier transform infrared spectroscopy (FTIR) spectra of the different agar samples were recorded using Perkin Elmer Spectrum Version 10.03.06 instrument. The samples were prepared in the form of pellets by grinding 20 mg of sample with 80 mg of KBr. The FTIR spectrum of each pellet was recorded in the frequency range from 400 cm-1 to 4000 cm-1. Tensile testing of the agar films was performed using Instron universal testing machine model no. 3345 with a load cell of 5 kN. ASTM (American society for testing and materials) D882-
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12 standard was used for testing of the films. Each film was cut into 10 rectangular strips of dimensions (70 mm × 10 mm × 0.050 mm) with 40 mm as gauge length following the above standard. Samples were stored at 50% RH for 48 hours before tensile testing to equilibrate the
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moisture in them. Strips were stretched at a rate of 10 mm/minute at 50% RH (relative humidity) as per the above standard.
Thermogravimetric analysis (TGA) of neat and crosslinked agar films was performed using
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Perkin Elmer STA (Simultaneous thermal analyzer) 8000 in the range 30°C-600°C at a heating
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rate of 10°C per minute under nitrogen gas.
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Hemocompatibility test
For the hemocompatibility assay, samples were washed thrice with phosphate buffered saline
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(1× PBS) and the assay was performed as mentioned elsewhere (Fischer, Li, Ahlemeyer, Krieglstein, & Kissel, 2003). The hemolysis percentage was calculated using the following
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formula:
𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠(%) =
𝐴𝑏𝑠𝑜𝑓𝑡𝑒𝑠𝑡𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑏𝑠𝑜𝑓𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 𝐴𝑏𝑠𝑜𝑓𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑓𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑐𝑜𝑛𝑡𝑟𝑜𝑙
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Where, Abs is the absorbance of the supernatant measured at 540 nm.
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Cytocompatibility test
For biocompatibility assay, NIH 3T3 [NCCS (National center for cell science)] cells were cultured in the high-glucose DMEM medium containing 10% FBS and penicillin-streptomycin
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(10000 U/mL) and incubated at 37°C in a humidified environment with 5% CO2. Meanwhile, samples were cut into 9 mm diameter circular disc and were sterilized by placing in gradient ethanol (70%, 90% and 100% ethanol) for 24 h. Samples were then washed thrice with 1× PBS and incubated with incomplete DMEM for 24 h in 48 well micro-titer plates. After 70% confluence, the cells were harvested using trypsin solution. 5×104 cells/well were seeded into 48 well plate and were further incubated with complete media for 7 days. 2D cell 5 | Page
culture on tissue culture plate (TCP) well was used as a positive control on which an equal number of cells were seeded. At predefined time intervals, DMEM media was removed from the well and replenished with 200 µL MTT solutions (0.5 mg/mL MTT in incomplete DMEM medium). After 4 h incubation, MTT was replaced by 500 µL DMSO and incubated for further 10 min under the same conditions. 100 µL solution from each well was transferred to a 96-well plate and the color change was measured at a wavelength of 570 nm using an ELISA (Enzyme
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linked immunosorbent assay) plate reader. Wettability: The contact angle of the films was measured with Dataphysics OCA35, (Germany). The wet contact angle was taken by immersing the films in PBS for four hours and
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removing the excess water by damping it with tissue paper. The measurement was carried out for three samples and the mean values of the contact angle measurement were calculated. 3.
Result and discussion
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3.1 Crosslinking reaction scheme and FTIR spectroscopy
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(b)
Figure 1. Crosslinking reaction scheme of agar by (a) 4, 4 diphenyl diisocyanate (DDI) and (b) 1, 6 hexamethylene diisocyanate (HDI). Crosslinking results in the formation of carbamate crosslinks between agar chains
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Crosslinking of agar by diisocyanates results in the formation of carbamate crosslinks between agar chains (Figure 1a and 1b). Figure 1 shows an example of intermolecular crosslinking where the crosslinker reacts with two separate chains of agar. Both ends of the crosslinker may react with the same chain resulting in intermolecular crosslinking. It is also possible to have only one end of crosslinker reacts with the polymer chain. In this manuscript we have considered intermolecular crosslinking.
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Figure 2 is FTIR spectra of agar and diisocyanate crosslinked agar. Crosslinking reduces the number of hydroxyl groups of agar chains and introduce carbamate crosslink bond. The short
dashed lines at 3300 cm-1 and 1567 cm-1 represents charcatetstic hydroxyl (–OH) and amide II
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(–NH–C=O–) bands in figure 2.
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Figure 2. FTIR spectra of 25% w/w 4, 4 Diphenyl diisocyanate (DDI) and 30% w/w 1, 6 Hexamethylene diisocyanate (HDI) crosslinked agar films. The characteristic amide (II) at
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1570 cm-1 is represented by short dashed lines. The absorbance band at 1567 cm-1 in crosslinked agar samples show presence of carbamate crosslink (amide (II) band) that confirms crosslinking of agar (Krumova, López, Benavente, Mijangos, & Pereña, 2000). The peak position of the characteristic bands is same for DDI, and HDI crosslinked agar samples.
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3.2 Swelling study - water uptake A large number of hydroxyl groups present on agar attract water molecules. The incoming water occupies space between agar chains by displacing them and hence cause swelling. The absorbed water also reduces the bonding between agar chains. Here, in crosslinked agar samples, crosslinking between hydroxyl (-OH) groups of agar and isocyanate (-N=C=O) groups of DI forms carbamate crosslinks, which decreases the number of hydroxyl groups. In
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addition, the crosslinks restrict movement of the polymer chains that further reduce swelling in crosslinked agar samples. Therefore, it can be expected to reduce swelling with increasing crosslinking. The number of crosslinks between agar chains is enhanced by increasing DI
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(crosslinker concentration) in crosslinked agar samples. From figure 3, it is observed that increasing crosslinker concentration in crosslinked agar samples successively reduce the
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swelling percentage and show water resistance.
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Figure 3. Swelling plot of 4, 4 Diphenyl diisocyanate (DDI) and 1, 6 Hexamethylene diisocyanate (HDI) crosslinked agar films. It is clearly observed from the figure that crosslinking gets saturated at 25% for DDI and 30% w/w for HDI, as there is faintly change in swelling percentage is observed. DDI crosslinked samples demonstrate a lower swelling percentage in comparison to HDI crosslinked samples (Table 1). The difference in swelling percentage observed between DDI 8 | Page
and HDI crosslinked agar could be due to difference in reactivity. Since isocyanate group has R-N=C=O sequence. On considering the resonating structures, the nitrogen is negatively charged and carbon is positively charged, therefore, if R, alkyl substituent attached to isocyanate group is aromatic, then the negative charge get delocalized into R, which makes aromatic diisocyanate (DDI) more reactive in comparison to aliphatic diisocyanate (HDI) (Sharmin & Zafar, 2012), hence it reacts with larger number of hydroxyl groups of agar.
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The swelling plots (figure 3) justify the above explanations. In figure 3, it is observed that swelling percentage decreases successively on increasing DDI concentration and after critical concentration of 25% w/w DDI, hardly any difference in swelling percentage is observed. It
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indicates the saturation in swelling resistance. Similar to the swelling trend of DDI, the
continuous decrease in the swelling percentage with increasing HDI concentration can be observed in the figure 3. However, the critical concentration for DDI is 25% w/w and for HDI
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is 35% w/w. Even at lower concentrations, DDI crosslinked agar restricts swelling more effectively in comparison to HDI, which is due to the reactivity difference between the
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crosslinkers as explained above.
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3.3 Tensile testing
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Figure 4a. Typical stress stain plot of (I) non-crosslinked agar, (II) 30% w/w 1, 4 diphenyl diisocyanate (DDI) and (III) 35% w/w 1, 6 hexamethylene diisocyanate (HDI) crosslinked agar. The plot represents the maximum strength obtained by each diisocyanate crosslinked film.
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(I)
(II)
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Figure 4b. Mechanical properties vs DI concentration (I) 4, 4 diphenyl diisocynate and (II)
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1, 6 hexamethylene diisocyanate (HDI) crosslinked agar The mechanical properties in terms of tensile strength and elongation at break for agar, DDI and HDI crosslinked agar are summarized in Table 1. Figure 4a represents typical stress-strain
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plot of agar, DDI and HDI crosslinked agar samples. Crosslinking of agar leads to the formation of large number of crosslinks that restricts the mobility of polymer chains and hence results in improved strength of the polymer with decrease in elongation at break. The mechanical properties also depend upon the geometry of the crosslinker (A. K. Sonker et al., 2016), which is observed in the case of two diisocyanates used in this study.
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The DDI crosslinked agar show 37% higher tensile strength and 8.9% less elongation at break in comparison to agar (figure 4b). The two rigid aromatic crosslinkers impart restriction to the mobility of polymer chains resulting in the observed properties. Unlike the aromatic crosslinker (DDI), HDI crosslinked agar shows lesser tensile strength and more elongation at break than agar. It could be due to the linear flexible structure of aliphatic diisocyanate. The tensile strength of crosslinked agar decreases at higher concentration of diisocyanates.
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There could be two opposing factors playing: an increase in crosslinking density with higher crosslinker concentration results in improvement of strength. Application of large amount of crosslinker, on the other hand, leads to unused diisocyanates in agar matrix, which upon
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washing may leave behind micropores causing reduction in strength. The swelling data also confirms that extent of crosslinking does not change at extreme diisocyanate concentrations. Swelling %
Tensile Strength (MPa)
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Agar films
31.7 (1.3)
13.8 (1.4)
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206 (6.6)*
Agar
Elongation at break (%)
132.7 (3.2)
28.8 (0.8)
9.5 (1.3)
10% w/w
101.4 (1.9)
35.7 (2.5)
9.5 (1.1)
72.7 (4)
37.7 (1.6)
5 (0.5)
55.6 (4.8)
43.9 (0.9)
6.2 (0.9)
34.6 (5)
43.5 (3.2)
4.9 (0.5)
33.2 (5.5)
45.3 (2.3)
3.8 (0.4)
34.4 (2)
32.6 (1.8)
2.6 (0.1)
33.6 (6.1)
34.4 (1.8)
3.4 (0.1)
5% w/w
135.7 (6.5)
20.5 (1.2)
15.8 (2.1)
10% w/w
92.7 (8.1)
22.4 (1.5)
22.7 (3.3)
15% w/w
75.2 (2.1)
28.7 (0.6)
18.7 (1.6)
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5% w/w
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DDI crosslinked Agar
15% w/w 20% w/w
30% w/w
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35% w/w
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25% w/w
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40% w/w
HDI crosslinked Agar
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20% w/w
67.5 (2.3)
25.9 (1.3)
13.1 (1.4)
25% w/w
61.7 (5.1)
30.6 (0.5)
9.5 (0.2)
30% w/w
51.4 (3.1)
28.2 (0.6)
14.4 (2)
35% w/w
46.7 (5.4)
30.2 (0.7)
12 (2.2)
40% w/w
46.3 (5.2)
27.1 (2)
15.8 (1)
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*Standard error of the mean values
Table 1. Swelling percentage and mechanical properties of agar, DDI and HDI crosslinked
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agar
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3.4 Thermal analysis
Figure 5. TGA thermogram of 25% w/w 4, 4 Diphenyl diisocyanate (DDI) and 30% w/w 1, 6
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Hexamethylene diisocyanate (HDI) crosslinked agar films. It is observed that thermal resistance of crosslinked samples is improved by crosslinking.
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Figure 6. DTGA thermogram of 25% w/w 4, 4 Diphenyl diisocyanate (DDI) and 30% w/w 1,
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6 Hexamethylene diisocyanate (HDI) crosslinked agar films. The crosslinked samples show
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slow rate of thermal degradation. The short dashed lines represent decomposition temperature
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of samples.
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Crosslinking of agar by diisocyanates improve its thermal stability at higher temperatures (figure 5). The TGA graphs of agar and crosslinked agar are analyzed in two regions. The first
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region from 50-200ºC shows weight loss in the agar and crosslinked agar samples due to the removal of moisture and weakly bound water molecules. All the samples, agar, DDI, and HDI crosslinked show similar weight loss in this region. The second stage, from 200-500ºC, corresponds to chain scission and degradation of agar
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backbone. In this stage, crosslinking stabilized the agar chains by providing strong inter and intra crosslink carbamate ester network. DDI crosslinked agar samples have retained 70-60%
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weight in this temperature range while agar has retained only 40% weight (figure 5). HDI crosslinked agar samples also demonstrate thermal resistance (figure 5), which is not as
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high as observed in the case of DDI crosslinked agar. This could be due to the difference in the reactivity of crosslinkers as DDI crosslinks more effectively, which in turn providing higher thermal stability than HDI. Moreover, at high temperatures aromatic diisocyanate containing polyurethane are thermally more stable than the aliphatic diisocyanates (Chattopadhyay, Sreedhar, & Raju, 2005).
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Differential thermogravimetric analysis (DTGA) plots of agar and crosslinked agar are also considered to check the rate of decomposition and shift in the decomposition temperature of agar by diisocyanate crosslinking. The DTGA plot (figure 6) of DDI crosslinked agar shows no shift in the decomposition temperature in comparison to agar (250°C). However, rate of decomposition is slow as DDI crosslinked agar becomes thermal resistant due to crosslinking. 3.5 Hemotoxicity study
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Hemolysis study was performed to determine the hemocompatibility of the crosslinked agar
samples. As can be observed from (figure 7) agar-diisocyanates samples show less than 5%
hemolysis. Therefore, since materials showing hemolysis less than 5 % are considered as
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hemocompatible (Autian, 1975), the crosslinked samples are also hemocompatible and can be
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utilized for applications where it needs to come in direct contact with blood.
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Figure 7. Hemocompatibility of crosslinked agar samples. Both the crosslinked samples show negative hemolysis result comparable to agar samples
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3.6 Cytocompatibility study
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Figure 8. Cytocompatibility of diisocyanate crosslinked agar samples. The results obtained
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confirmed that crosslinked agar samples are favorable for cell proliferation
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MTT method is widely used for the preliminary biocompatibility evaluation of the biomaterials (Mansur, de S. Costa, Mansur, & Barbosa-Stancioli, 2009). Therefore, cell compatibility of
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crosslinked agar was also determined using MTT assay. Figure 8 shows that for fibroblast (NIH 3T3) cells, cross linked samples show cell viability in the range of 65% to 85% compared
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to positive control (TCP). This decrease in cell viability could be correlated with surface wettability that plays an important role in cell adhesion and proliferation (Bumgardner et al., 2003; Good, 1992); with a water contact angle between 40° and 70° promoting cell adhesion
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(Massoumi, Aali, & Jaymand, 2015).
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Figure 9. Contact angle measurements of agar and crosslinked agar samples
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Our crosslinked samples had a water contact angle of ~70° for 15% w/v HDI crosslinked
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samples and ~90° for 15% w/v DDI crosslinked samples (Figure 9). Since these contact angles
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are at the higher end of optimum contact angle for cell adhesion, it therefore explains the low cytocompatibility. Besides poor adhesion of cells on the samples, the lower viability could also
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be attributed to the toxicity of degradation products of crosslinking (Barrioni, de Carvalho, Oréfice, de Oliveira, & Pereira, 2015). 4.
Conclusion
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In this work we demonstrate that crosslinking of agar using DDI (4, 4 diphenyl diisocyanate) (DDI) and HDI (1, 6 hexamethylene diisocyanate) improves the mechanical and thermal
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properties of agar with a noticeable reduction in the water uptake. The efficacy of aromatic and aliphatic diisocyanate cross linking on agar properties is also different. Agar crosslinked with aromatic diisocyanates showed higher strength and thermal resistance than aliphatic
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crosslinked agar, which could be attributed to the higher reactivity and rigidity of the aromatic cross-linker. The crosslinked agar films showed positive hemocompatibility and moderate cytocompatibility. The diisocyanate crosslinked agar films could thus find promising applications in biodegradable packaging and biomedical applications. Conflict of interest
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Authors have filed an Indian patent with application numbered 201811002306 dated January 19, 2018. The work reported here is part of this patent. Acknowledgements Authors like to thank, Department of Science and Technology – Technology Systems Development Programme (DST/TSG/AMT/2015/329), Government of India for financial support. Author thanks Prof. S Ganesh, Department of Biological Sciences and Bioengineering
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for ELISA facility, Prof. Krishnacharya Khare, Department of Physics at IIT Kanpur and for providing access to contact angle measurement facility respectively. Authors also thanks
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BIRAC-bioincubator (SIDBI, IITK) for access to cell culture facility.
References Atef, M., Rezaei, M., & Behrooz, R. (2014). Preparation and characterization agar-based nanocomposite film reinforced by nanocrystalline cellulose. International Journal of Biological Macromolecules, 70, 537-544.
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Derived Clay Nanocomposite Film. Journal of Food Science, 77(12), N66-N73. Rhim, J.-W., & Ng, P. K. W. (2007). Natural Biopolymer-Based Nanocomposite Films for
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S0144-8617(18)31057-9 https://doi.org/10.1016/j.carbpol.2018.08.138 CARP 14035
To appear in: Received date: Revised date: Accepted date:
7-4-2018 27-8-2018 30-8-2018
Please cite this article as: Sonker AK, Belay M, Rathore K, Jahan K, Verma S, Ramanathan G, Verma V, Crosslinking of agar by diisocyanates, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.08.138 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Crosslinking of agar by diisocyanates
Amit Kumar Sonker1, Mezigebu Belay1, Kalpana Rathore1, Kousar Jahan1, Sankalp Verma1, Gurunath Ramanathan2, Vivek Verma1, 3 1
Department of Materials Science and Engineering, Indian Institute of Technology Kanpur,
2
3
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Kanpur - 208016 Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur - 208016
Centre of Environmental Science and Engineering, Indian Institute of Technology Kanpur,
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Kanpur - 208016
Corresponding author’s email: [email protected]
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Highlights
Agar based bioplastic is prepared by chemical crosslinking with diisocyanates
Efficacy of DDI (4, 4 diphenyl diisocyanate) and HDI (1, 6 hexamethylene
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N
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diisocyanate) is compared on agar crosslinking
Crosslinked agar films show improved tensile strength and water resistance
Crosslinked films are non-toxic to blood and show cell proliferation
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Abstract
In the present study, crosslinking of agar using diisocyanate (DI) was demonstrated to limit the
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high water absorption property of agar. In addition, the efficacy of aromatic diisocyanate, DDI (4, 4 diphenyl diisocyanate) and aliphatic diisocyanate, HDI (1, 6 hexamethylene diisocyanate) on crosslinked agar properties was compared. The water uptake was successfully reduced by
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crosslinking and its minimum values observed for DDI and HDI crosslinked agar was 33.6% and 43.6%, respectively in comparison to agar (206%). The maximum tensile strength was observed for DDI crosslinked agar (45.3 MPa) which was higher than HDI crosslinked agar (30.6 MPa) and agar (31.7 MPa). The aromatic diisocyanates crosslinked agar showed better thermal resistance at higher temperature. It was observed that aromatic diisocyanate crosslinked agar more effectively than the aliphatic diisocyanate due to the higher reactivity. 1 | Page
The crosslinked agar samples were hemocompatible and show non-toxic nature for cell proliferation. Keywords:
bioplastic,
crosslinking,
agar,
cytocompatibility,
hemocompatibility.
Abbreviations DI – Diisocyanate
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DDI – 4, 4 Diphenyl Diisocyanate
1.
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HDI – 1, 6 Hexamethylene Diisocyanate
Introduction
Synthetic polymers such as low-density polyethylene, high-density polyethylene,
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polypropylene, polyethylene terephthalate, polystyrene are widely used packaging material.
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However, increasing demand and consumption of these ‘nondegradable’ polymers is promoting a negative impact on our ecosystem, which is necessitating thrust to develop their
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biodegradable counterparts with comparable properties (Bordes, Pollet, & Avérous, 2009; J.-
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W. Rhim, Park, & Ha, 2013; Shit & Shah, 2014; Siracusa, Rocculi, Romani, & Rosa, 2008; Tang, Kumar, Alavi, & Sandeep, 2012; Weber, Haugaard, Festersen, & Bertelsen, 2002).
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Among biodegradable polymers like polylactic acid, starch, cellulose etc., a considerable interest is developed in agar as potential packaging material (J. P. Lee, K. H. Lee, & H. K. Song, 1997; López de Lacey, López-Caballero, & Montero, 2014; Orsuwan, Shankar, Wang, Sothornvit, & Rhim, 2016; Phan The, Debeaufort, Voilley, & Luu, 2009; J.-W. Rhim & Ng,
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2007; Shankar, Reddy, & Rhim, 2015; Shankar & Rhim, 2016; Siracusa et al., 2008; Weber et al., 2002). Agar is a natural, biodegradable, water soluble and cost-effective polymer (Freile-
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Pelegrín et al., 2007; J. P. Lee et al., 1997; Skurtys et al., 2010). It is a polysaccharide extracted from agrophyte algae that belong to the phylum of Rhodophyta, which is found in seaweed
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(McHugh, 1987). Since it is extracted from seaweed, the use of agar as a packaging material neither cause deforestation, like cellulose, nor affect food supply chain, like starch. It is a mixture of gelling fraction agarose and non-gelling fraction agaropectin, which is sulfated and slightly branched (Phillips & Williams, 2000). Potentially, agar as packaging materials is reported earlier (Kanmani & Rhim, 2014; J.-P. LEE, K.-H. LEE, & H.-K. SONG, 1997; Phan The et al., 2009; J.-W. Rhim, 2011, 2012; J. W. Rhim, Wang, & Hong, 2013). However, its high water absorption, moderate mechanical strength, and thermal stability limit its application 2 | Page
as biodegradable packaging material. To overcome the above drawbacks, reinforcement and chemical cross linking of agar has been attempted earlier (Atef, Rezaei, & Behrooz, 2014; Awadhiya, Kumar, Rathore, Fatma, & Verma, 2016; Orsuwan et al., 2016; J.-W. Rhim, 2011; J. W. Rhim et al., 2013; Shankar et al., 2015; Shankar & Rhim, 2016). For example, Awadhiya et al. (Awadhiya, Kumar, Rathore, et al., 2016) showed change of tensile strength from 25.1 MPa for pure agarose to 60.1 MPa for agarose/bacterial cellulose composite. Shankar et al. (Shankar et al., 2015) reported an improvement of tensile strength from 45.7 MPa for pure agar
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to 52.1 MPa for lignin/agar composites. Rhim et al. (J.-W. Rhim, 2012) showed an increase in tensile strength of agar (31 MPa) for pure agar to 45 MPa for agar/ κ-carrageenan composites.
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Awadhiya et al. (Awadhiya, Kumar, & Verma, 2016) showed a reduction in water uptake by ~8 fold with improvement in mechanical and thermal properties by crosslinking agarose with citric acid. However, higher concentration of citric acid deteriorated the mechanical properties
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of agarose due to the hydrolyzing role of acid on agarose chains during crosslinking at high temperature. To overcome this, a process to crosslink agar is proposed that does not promote
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chain degradation during crosslinking.
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Diisocyanates do not promote chain scission and hence can also be used in higher
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concentration. Moreover, high temperature for crosslinking is not required in case of diisocyanates. It is also reasonable to expect the role of crosslinker geometry on the property
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of polymer (Sonker, Rathore, Nagarale, & Verma, 2017; Sonker, Tiwari, Nagarale, & Verma, 2016). Therefore, the aim of this work is to crosslink agar by diisocyanates to reduce its water absorption in addition to and improving the tensile strength and thermal stability. Further, the efficacy of aromatic, DDI (4, 4 diphenyl diisocyanate) and aliphatic diisocyanate, HDI (1, 6
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hexamethylene diisocyanate) on agar crosslinking is compared. For suitable comparison, swelling, mechanical and thermal properties of diisocyanates crosslinked agar are discussed in
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detail. Moreover, hemocompatibility and cytocompatibility of agar and crosslinked agar are studied for potential applications in biomedical field. Material and methods
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2.
2.1 Materials Agar (gelling temperature 34-36°C, pH 6.0-7.0, dissolution in water at 85°C) was purchased from Hi Media, India. 4, 4 diphenyl diisocyanate (DDI) and hexamethylene diisocyanate (HDI) were procured from Sigma Aldrich, India. Dimethylsulphoxide (DMSO) and tetrahydrofuran (THF) were purchased from Merck, Germany. Polypropylene (PP) petridishes were purchased 3 | Page
from Tarsons Product Pvt. Ltd. For phosphate buffered saline (PBS) solution, potassium chloride, sodium chloride, potassium dihydrogen orthophosphate, disodium hydrogen orthophosphate were purchased from Thermo Fisher Scientific (Mumbai), India. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was procured from Sisco Research Laboratories (SRL) Pvt. Ltd, India. Dulbecco’s Modified Eagle’s medium-high glucose (DMEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (EDTA) and antibiotic (penicillin and streptomycin) solution were purchased from Gibco, Thermo
2.2 Preparation of agar and diisocyanates crosslinked films
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Fisher, India. Ethanol was purchased from Merck specialties Private Limited (Mumbai).
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Agar (1 g) was dissolved in 40 mL DMSO at 80°C. The solution was then cooled down from 80°C to 10°C using an ice bath. For crosslinking, the concentrations of DDI and HDI were varied from 5% to 40% w/w (with respect to agar) with an interval of 5%. The required amount
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of crosslinker (DDI or HDI) was dissolved in 10 mL of DMSO at 10°C. The agar solution (40
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mL) was transferred into crosslinker solution (10 mL) and was mixed vigorously using a magnetic stirrer for 10-15 minutes. The solution was then poured into polypropylene petridish
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and allowed to react for 12 hours at room temperature. The petridish was kept in hot air oven
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at 60°C for 36 hours to remove DMSO. The thickness of the dried film was ~50 micron as measured using Mitutoyo micrometer. The crosslinked agar films were washed with THF to
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remove unreacted diisocyanate. Washing was performed using a mechanical shaker for 12 hours and then the films were dried at 60°C to remove THF. 2.3 Characterization of films
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Swelling study was performed to measure the amount of absorbed water according to the ISO (International Organization of Standardization) standard (ISO 62:2008, Plastics 2
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Determination of water absorption). Agar films were cut into three square pieces (2 cm
cm), weighed and immersed in deionized water for 24 hours at 25°C. The swollen samples were taken out and wiped with tissue paper to remove excess water from the film surface and
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weighed. The swelling percentage in agar and diisocyanate crosslinked agar samples is calculated by using equation (1) (1)
where WS is the weight of swollen samples and WD is the weight of dry samples. 4 | Page
Fourier transform infrared spectroscopy (FTIR) spectra of the different agar samples were recorded using Perkin Elmer Spectrum Version 10.03.06 instrument. The samples were prepared in the form of pellets by grinding 20 mg of sample with 80 mg of KBr. The FTIR spectrum of each pellet was recorded in the frequency range from 400 cm-1 to 4000 cm-1. Tensile testing of the agar films was performed using Instron universal testing machine model no. 3345 with a load cell of 5 kN. ASTM (American society for testing and materials) D882-
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12 standard was used for testing of the films. Each film was cut into 10 rectangular strips of dimensions (70 mm × 10 mm × 0.050 mm) with 40 mm as gauge length following the above standard. Samples were stored at 50% RH for 48 hours before tensile testing to equilibrate the
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moisture in them. Strips were stretched at a rate of 10 mm/minute at 50% RH (relative humidity) as per the above standard.
Thermogravimetric analysis (TGA) of neat and crosslinked agar films was performed using
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Perkin Elmer STA (Simultaneous thermal analyzer) 8000 in the range 30°C-600°C at a heating
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rate of 10°C per minute under nitrogen gas.
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Hemocompatibility test
For the hemocompatibility assay, samples were washed thrice with phosphate buffered saline
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(1× PBS) and the assay was performed as mentioned elsewhere (Fischer, Li, Ahlemeyer, Krieglstein, & Kissel, 2003). The hemolysis percentage was calculated using the following
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formula:
𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠(%) =
𝐴𝑏𝑠𝑜𝑓𝑡𝑒𝑠𝑡𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑏𝑠𝑜𝑓𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 𝐴𝑏𝑠𝑜𝑓𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑓𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑐𝑜𝑛𝑡𝑟𝑜𝑙
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Where, Abs is the absorbance of the supernatant measured at 540 nm.
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Cytocompatibility test
For biocompatibility assay, NIH 3T3 [NCCS (National center for cell science)] cells were cultured in the high-glucose DMEM medium containing 10% FBS and penicillin-streptomycin
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(10000 U/mL) and incubated at 37°C in a humidified environment with 5% CO2. Meanwhile, samples were cut into 9 mm diameter circular disc and were sterilized by placing in gradient ethanol (70%, 90% and 100% ethanol) for 24 h. Samples were then washed thrice with 1× PBS and incubated with incomplete DMEM for 24 h in 48 well micro-titer plates. After 70% confluence, the cells were harvested using trypsin solution. 5×104 cells/well were seeded into 48 well plate and were further incubated with complete media for 7 days. 2D cell 5 | Page
culture on tissue culture plate (TCP) well was used as a positive control on which an equal number of cells were seeded. At predefined time intervals, DMEM media was removed from the well and replenished with 200 µL MTT solutions (0.5 mg/mL MTT in incomplete DMEM medium). After 4 h incubation, MTT was replaced by 500 µL DMSO and incubated for further 10 min under the same conditions. 100 µL solution from each well was transferred to a 96-well plate and the color change was measured at a wavelength of 570 nm using an ELISA (Enzyme
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linked immunosorbent assay) plate reader. Wettability: The contact angle of the films was measured with Dataphysics OCA35, (Germany). The wet contact angle was taken by immersing the films in PBS for four hours and
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removing the excess water by damping it with tissue paper. The measurement was carried out for three samples and the mean values of the contact angle measurement were calculated. 3.
Result and discussion
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3.1 Crosslinking reaction scheme and FTIR spectroscopy
(a)
(b)
Figure 1. Crosslinking reaction scheme of agar by (a) 4, 4 diphenyl diisocyanate (DDI) and (b) 1, 6 hexamethylene diisocyanate (HDI). Crosslinking results in the formation of carbamate crosslinks between agar chains
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Crosslinking of agar by diisocyanates results in the formation of carbamate crosslinks between agar chains (Figure 1a and 1b). Figure 1 shows an example of intermolecular crosslinking where the crosslinker reacts with two separate chains of agar. Both ends of the crosslinker may react with the same chain resulting in intermolecular crosslinking. It is also possible to have only one end of crosslinker reacts with the polymer chain. In this manuscript we have considered intermolecular crosslinking.
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Figure 2 is FTIR spectra of agar and diisocyanate crosslinked agar. Crosslinking reduces the number of hydroxyl groups of agar chains and introduce carbamate crosslink bond. The short
dashed lines at 3300 cm-1 and 1567 cm-1 represents charcatetstic hydroxyl (–OH) and amide II
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(–NH–C=O–) bands in figure 2.
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Figure 2. FTIR spectra of 25% w/w 4, 4 Diphenyl diisocyanate (DDI) and 30% w/w 1, 6 Hexamethylene diisocyanate (HDI) crosslinked agar films. The characteristic amide (II) at
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1570 cm-1 is represented by short dashed lines. The absorbance band at 1567 cm-1 in crosslinked agar samples show presence of carbamate crosslink (amide (II) band) that confirms crosslinking of agar (Krumova, López, Benavente, Mijangos, & Pereña, 2000). The peak position of the characteristic bands is same for DDI, and HDI crosslinked agar samples.
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3.2 Swelling study - water uptake A large number of hydroxyl groups present on agar attract water molecules. The incoming water occupies space between agar chains by displacing them and hence cause swelling. The absorbed water also reduces the bonding between agar chains. Here, in crosslinked agar samples, crosslinking between hydroxyl (-OH) groups of agar and isocyanate (-N=C=O) groups of DI forms carbamate crosslinks, which decreases the number of hydroxyl groups. In
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addition, the crosslinks restrict movement of the polymer chains that further reduce swelling in crosslinked agar samples. Therefore, it can be expected to reduce swelling with increasing crosslinking. The number of crosslinks between agar chains is enhanced by increasing DI
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(crosslinker concentration) in crosslinked agar samples. From figure 3, it is observed that increasing crosslinker concentration in crosslinked agar samples successively reduce the
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swelling percentage and show water resistance.
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Figure 3. Swelling plot of 4, 4 Diphenyl diisocyanate (DDI) and 1, 6 Hexamethylene diisocyanate (HDI) crosslinked agar films. It is clearly observed from the figure that crosslinking gets saturated at 25% for DDI and 30% w/w for HDI, as there is faintly change in swelling percentage is observed. DDI crosslinked samples demonstrate a lower swelling percentage in comparison to HDI crosslinked samples (Table 1). The difference in swelling percentage observed between DDI 8 | Page
and HDI crosslinked agar could be due to difference in reactivity. Since isocyanate group has R-N=C=O sequence. On considering the resonating structures, the nitrogen is negatively charged and carbon is positively charged, therefore, if R, alkyl substituent attached to isocyanate group is aromatic, then the negative charge get delocalized into R, which makes aromatic diisocyanate (DDI) more reactive in comparison to aliphatic diisocyanate (HDI) (Sharmin & Zafar, 2012), hence it reacts with larger number of hydroxyl groups of agar.
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The swelling plots (figure 3) justify the above explanations. In figure 3, it is observed that swelling percentage decreases successively on increasing DDI concentration and after critical concentration of 25% w/w DDI, hardly any difference in swelling percentage is observed. It
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indicates the saturation in swelling resistance. Similar to the swelling trend of DDI, the
continuous decrease in the swelling percentage with increasing HDI concentration can be observed in the figure 3. However, the critical concentration for DDI is 25% w/w and for HDI
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is 35% w/w. Even at lower concentrations, DDI crosslinked agar restricts swelling more effectively in comparison to HDI, which is due to the reactivity difference between the
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crosslinkers as explained above.
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3.3 Tensile testing
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Figure 4a. Typical stress stain plot of (I) non-crosslinked agar, (II) 30% w/w 1, 4 diphenyl diisocyanate (DDI) and (III) 35% w/w 1, 6 hexamethylene diisocyanate (HDI) crosslinked agar. The plot represents the maximum strength obtained by each diisocyanate crosslinked film.
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(I)
(II)
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Figure 4b. Mechanical properties vs DI concentration (I) 4, 4 diphenyl diisocynate and (II)
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1, 6 hexamethylene diisocyanate (HDI) crosslinked agar The mechanical properties in terms of tensile strength and elongation at break for agar, DDI and HDI crosslinked agar are summarized in Table 1. Figure 4a represents typical stress-strain
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plot of agar, DDI and HDI crosslinked agar samples. Crosslinking of agar leads to the formation of large number of crosslinks that restricts the mobility of polymer chains and hence results in improved strength of the polymer with decrease in elongation at break. The mechanical properties also depend upon the geometry of the crosslinker (A. K. Sonker et al., 2016), which is observed in the case of two diisocyanates used in this study.
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The DDI crosslinked agar show 37% higher tensile strength and 8.9% less elongation at break in comparison to agar (figure 4b). The two rigid aromatic crosslinkers impart restriction to the mobility of polymer chains resulting in the observed properties. Unlike the aromatic crosslinker (DDI), HDI crosslinked agar shows lesser tensile strength and more elongation at break than agar. It could be due to the linear flexible structure of aliphatic diisocyanate. The tensile strength of crosslinked agar decreases at higher concentration of diisocyanates.
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There could be two opposing factors playing: an increase in crosslinking density with higher crosslinker concentration results in improvement of strength. Application of large amount of crosslinker, on the other hand, leads to unused diisocyanates in agar matrix, which upon
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washing may leave behind micropores causing reduction in strength. The swelling data also confirms that extent of crosslinking does not change at extreme diisocyanate concentrations. Swelling %
Tensile Strength (MPa)
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Agar films
31.7 (1.3)
13.8 (1.4)
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206 (6.6)*
Agar
Elongation at break (%)
132.7 (3.2)
28.8 (0.8)
9.5 (1.3)
10% w/w
101.4 (1.9)
35.7 (2.5)
9.5 (1.1)
72.7 (4)
37.7 (1.6)
5 (0.5)
55.6 (4.8)
43.9 (0.9)
6.2 (0.9)
34.6 (5)
43.5 (3.2)
4.9 (0.5)
33.2 (5.5)
45.3 (2.3)
3.8 (0.4)
34.4 (2)
32.6 (1.8)
2.6 (0.1)
33.6 (6.1)
34.4 (1.8)
3.4 (0.1)
5% w/w
135.7 (6.5)
20.5 (1.2)
15.8 (2.1)
10% w/w
92.7 (8.1)
22.4 (1.5)
22.7 (3.3)
15% w/w
75.2 (2.1)
28.7 (0.6)
18.7 (1.6)
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5% w/w
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DDI crosslinked Agar
15% w/w 20% w/w
30% w/w
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35% w/w
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25% w/w
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40% w/w
HDI crosslinked Agar
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20% w/w
67.5 (2.3)
25.9 (1.3)
13.1 (1.4)
25% w/w
61.7 (5.1)
30.6 (0.5)
9.5 (0.2)
30% w/w
51.4 (3.1)
28.2 (0.6)
14.4 (2)
35% w/w
46.7 (5.4)
30.2 (0.7)
12 (2.2)
40% w/w
46.3 (5.2)
27.1 (2)
15.8 (1)
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*Standard error of the mean values
Table 1. Swelling percentage and mechanical properties of agar, DDI and HDI crosslinked
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agar
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3.4 Thermal analysis
Figure 5. TGA thermogram of 25% w/w 4, 4 Diphenyl diisocyanate (DDI) and 30% w/w 1, 6
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Hexamethylene diisocyanate (HDI) crosslinked agar films. It is observed that thermal resistance of crosslinked samples is improved by crosslinking.
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Figure 6. DTGA thermogram of 25% w/w 4, 4 Diphenyl diisocyanate (DDI) and 30% w/w 1,
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6 Hexamethylene diisocyanate (HDI) crosslinked agar films. The crosslinked samples show
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slow rate of thermal degradation. The short dashed lines represent decomposition temperature
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of samples.
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Crosslinking of agar by diisocyanates improve its thermal stability at higher temperatures (figure 5). The TGA graphs of agar and crosslinked agar are analyzed in two regions. The first
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region from 50-200ºC shows weight loss in the agar and crosslinked agar samples due to the removal of moisture and weakly bound water molecules. All the samples, agar, DDI, and HDI crosslinked show similar weight loss in this region. The second stage, from 200-500ºC, corresponds to chain scission and degradation of agar
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backbone. In this stage, crosslinking stabilized the agar chains by providing strong inter and intra crosslink carbamate ester network. DDI crosslinked agar samples have retained 70-60%
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weight in this temperature range while agar has retained only 40% weight (figure 5). HDI crosslinked agar samples also demonstrate thermal resistance (figure 5), which is not as
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high as observed in the case of DDI crosslinked agar. This could be due to the difference in the reactivity of crosslinkers as DDI crosslinks more effectively, which in turn providing higher thermal stability than HDI. Moreover, at high temperatures aromatic diisocyanate containing polyurethane are thermally more stable than the aliphatic diisocyanates (Chattopadhyay, Sreedhar, & Raju, 2005).
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Differential thermogravimetric analysis (DTGA) plots of agar and crosslinked agar are also considered to check the rate of decomposition and shift in the decomposition temperature of agar by diisocyanate crosslinking. The DTGA plot (figure 6) of DDI crosslinked agar shows no shift in the decomposition temperature in comparison to agar (250°C). However, rate of decomposition is slow as DDI crosslinked agar becomes thermal resistant due to crosslinking. 3.5 Hemotoxicity study
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Hemolysis study was performed to determine the hemocompatibility of the crosslinked agar
samples. As can be observed from (figure 7) agar-diisocyanates samples show less than 5%
hemolysis. Therefore, since materials showing hemolysis less than 5 % are considered as
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hemocompatible (Autian, 1975), the crosslinked samples are also hemocompatible and can be
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utilized for applications where it needs to come in direct contact with blood.
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Figure 7. Hemocompatibility of crosslinked agar samples. Both the crosslinked samples show negative hemolysis result comparable to agar samples
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3.6 Cytocompatibility study
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Figure 8. Cytocompatibility of diisocyanate crosslinked agar samples. The results obtained
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confirmed that crosslinked agar samples are favorable for cell proliferation
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MTT method is widely used for the preliminary biocompatibility evaluation of the biomaterials (Mansur, de S. Costa, Mansur, & Barbosa-Stancioli, 2009). Therefore, cell compatibility of
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crosslinked agar was also determined using MTT assay. Figure 8 shows that for fibroblast (NIH 3T3) cells, cross linked samples show cell viability in the range of 65% to 85% compared
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to positive control (TCP). This decrease in cell viability could be correlated with surface wettability that plays an important role in cell adhesion and proliferation (Bumgardner et al., 2003; Good, 1992); with a water contact angle between 40° and 70° promoting cell adhesion
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(Massoumi, Aali, & Jaymand, 2015).
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Figure 9. Contact angle measurements of agar and crosslinked agar samples
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Our crosslinked samples had a water contact angle of ~70° for 15% w/v HDI crosslinked
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samples and ~90° for 15% w/v DDI crosslinked samples (Figure 9). Since these contact angles
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are at the higher end of optimum contact angle for cell adhesion, it therefore explains the low cytocompatibility. Besides poor adhesion of cells on the samples, the lower viability could also
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be attributed to the toxicity of degradation products of crosslinking (Barrioni, de Carvalho, Oréfice, de Oliveira, & Pereira, 2015). 4.
Conclusion
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In this work we demonstrate that crosslinking of agar using DDI (4, 4 diphenyl diisocyanate) (DDI) and HDI (1, 6 hexamethylene diisocyanate) improves the mechanical and thermal
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properties of agar with a noticeable reduction in the water uptake. The efficacy of aromatic and aliphatic diisocyanate cross linking on agar properties is also different. Agar crosslinked with aromatic diisocyanates showed higher strength and thermal resistance than aliphatic
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crosslinked agar, which could be attributed to the higher reactivity and rigidity of the aromatic cross-linker. The crosslinked agar films showed positive hemocompatibility and moderate cytocompatibility. The diisocyanate crosslinked agar films could thus find promising applications in biodegradable packaging and biomedical applications. Conflict of interest
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Authors have filed an Indian patent with application numbered 201811002306 dated January 19, 2018. The work reported here is part of this patent. Acknowledgements Authors like to thank, Department of Science and Technology – Technology Systems Development Programme (DST/TSG/AMT/2015/329), Government of India for financial support. Author thanks Prof. S Ganesh, Department of Biological Sciences and Bioengineering
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for ELISA facility, Prof. Krishnacharya Khare, Department of Physics at IIT Kanpur and for providing access to contact angle measurement facility respectively. Authors also thanks
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BIRAC-bioincubator (SIDBI, IITK) for access to cell culture facility.
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