The present invention relates to a process for the preparation of bioplastics. More particularly, the invention relates to a development of polysaccharides based bioplastics by crosslinking with diisocyanates and applications thereof.
BACKGROUND OF THE INVENTION
Synthetic polymers such as low-density polyethylene, high-density polyethylene, polypropylene, polyethylene terephthalate, polystyrene are widely used packaging material due to their availability and cost-effectiveness. However, increasing demand and consumption of these 'nondegradable' polymers is promoting a negative impact on our ecosystem, which is necessitating thrust to develop their biodegradable counterparts with comparable properties. Owing to these, there is a need to develop an alternative bioplastic material that can be derived from abundant resources (P. Bordes, et al., Progress in Polymer Science, 2009, 34, 125-155; C. J. Weber, et al., Food Additives & Contaminants, 2002, 19, 172-177; V. Siracusa, et al, Trends in Food Science & Technology, 2008, 19, 634-643; S. C. Shit et al, Journal of Polymers, 2014, 2014, 13; J.-W. Rhim, et al, Progress in Polymer Science, 2013, 38, 1629-1652; X. Z. Tang et al, Critical Reviews in Food Science and Nutrition, 2012, 52, 426-442).
Among biodegradable polymers like polylactic acid, starch, cellulose etc. a considerable interest is developed in agar as potential packaging material (Rhim et al., Critical Reviews in Food Science and Nutrition 47(4) (2007) 411-43, Shankar et al., International Journal of Biological Macromolecules 81 (2015) 267-273. Shankar et al., Carbohydrate Polymers 135 (2016) 18-26, J. Weber, et al., Food Additives & Contaminants, 2002, 19, 172-177; V. Siracusa, et al, Trends in Food Science & Technology, 2008, 19, 634-643). It is a natural, biodegradable, water soluble and cost-effective polymer (J. P. Lee, et al, Journal of Materials Science, 1997, 32, 5825-5832; Y. Freile-Pelegrin, Polymer Degradation and Stability, 2007, 92, 244-252; O. Skurtys,

C. et al., in Food Hydrocolloids: Characteristics, Properties and Structures, 2010, pp. 41-80). This polysaccharide is extracted from agrophyte algae that belong to the phylum of Rhodophyta, which is found in seaweed (D. J. McHugh, Production and utilization of products from commercial seaweeds, Food and Agriculture Organization of the United Nations, Rome, 1987). Since it is extracted from sea wood, the use of agar as a packaging material neither cause deforestation nor affect food supply. It is a mixture of gelling fraction agarose and non-gelling fraction agaropectin, which is sulfated and slightly branched (G. O. Phillips and P. A. Williams, Handbook of Hydrocolloids, CRC Press, 2000). This polysaccharide has potential application as packaging materials which is reported earlier. However, its mechanical strength, thermal stability, and water resistance are needed to be improved for its application as bioplastic material.
The reinforcement (nano-cellulose, clay etc.) and chemical crosslinking of agar have been done to make it usable as packaging plastic materials with desired properties. For example, Atef et al. International Journal of Biological Macromolecules, 2014, 70, 537-544, showed the change of tensile strength from 18.2 MPa for pure agar to 22.8 MPa for agar/ nanocrystalline cellulose composite with reduced water uptake ratio. Shankar et al. International Journal of Biological Macromolecules, 2015, 81, 267-273 reported an improvement of tensile strength from 45.7MPa for pure agar to 52.1 MPA for lignin/agar composites. Rhim et al. Journal of Food Science, 2012, 77, N66-N73, showed an increment of tensile strength from 31.03 MPa for pure agar to 45 MPa for agar/ K-Carrageenan composites.
In another report, Rhim et al, Food Hydrocolloids, 2013, 33, 327-335, prepared agar/silver nanoparticles composite and reported a change of tensile strength from 46.38 MPa for pure agar to 53.44 MPa for agar/silver composites and a change of contact angle from 49.94 to 84.68. Moreover, Rhim et al, Carbohydrate Polymers, 2011, 86, 691-699, showed an increment of tensile strength from 28.04 for pure agar to 36.87 for agar/clay composite. Kanmani et al, Carbohydrate Polymers, 2014, 102, 708-716 prepared agar/ grapefruit seed extract and showed high UV barrier property and improved antibacterial activity of the composite but with a decrease of tensile strength and thermal stability. Orsuwan et al., YoodHydrocolloids, 2016, 60, 476-485 prepared

agar/banana blend films reinforced with silver nanoparticles and reported improved the antibacterial property, water resistance and high UV screening property of the composite. Shukla et al Bioresource Technology, 2012, 107, 295-300; also prepared agar/silver nanoparticles based nanocomposite film with the antibacterial application.
Juhani et al. Bioresource Technology, 2012, 107, 295-300, demonstrated the crosslinking of agar with bifunctional groups (-COC1, -SO2CI, and -N=C=S). S. Hjerten, Journal, 1986, also showed the crosslinking of agar with alkaline surrounding (pH>ll) using divinylsulfone. Awadhiya et al, Carbohydrate Polymers, 2016, 151, 60-67 showed an improvement of tensile from 25.1MPa to 52.7 MPa with reduction of water absorption and improved thermal stability by crosslinking agarose with citric acid. However, higher concentration of citric acid deteriorated the mechanical properties 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 chain degradation during crosslinking.
Diisocyanates do not promote chain scission and hence they can also be used in higher 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 of polymer. Therefore, the aim of this work is to crosslink agar by diisocyanates to reduce its water absorption and improve tensile strength and thermal stability. Further, the efficacy of aromatic, DDI (4, 4 diphenyl diisocyanate) and aliphatic diisocyanate, HDI (1, 6 hexamethylene diisocyanate on agar crosslinking is compared. For suitable comparison, swelling, mechanical and thermal properties of diisocyanates crosslinked agar is discussed in detail.
US5494940A discloses a completely crosslinked, highly porous body derived from a water-soluble hydrogel polymer, selected from the group consisting of alginates, gums, starch, dextrin, agar, gelatins, casein, collagen, polyvinyl alcohol, polyethyleneimine, acrylate polymers, starch/acrylate copolymers, and mixtures and copolymers thereof. Dl teaches that porous body can be crosslinked with a diisocyanate by solvent exchange process involving replacing solvent in a polymer gel with another solvent.

On the contrary present invention uses solvent casting method for forming polymer samples in which both the polymer and crosslinker are mixed simultaneously and poured into the mold. The present method is simpler and faster to perform.
Patil, Sachin: Crosslinking of Polysaccharides: methods and Applications; Latest Reviews 6.2 (2008)] discloses various chemical crosslinking methods of polysaccharide which are highly versatile methods providing good mechanical stability. It mentions that polysaccharides can be crosslinked with 1, 6-hexa-methylene diisocyanate which forms network properties that can be easily tailored by the concentration of the dissolved polysaccharide and the amount of crosslinking agent. In this prior art, the crosslinking of polysaccharides by using various crosslinking methods is theoretically explained. However, the effect of crosslinking on water absorption, thermal and mechanical properties has yet not been reported. In addition, the method is reported for pharmaceutical application only.
Hirohito Yamasaki et. al.: J Chem Technol Biotechnol 83:991-997 (2008)] discloses crosslinked P-CyD prepolymer was synthesized by treatment of P-CyD with hexamethylene diisocyanate (HDI) at a molar ratio of 1:8. Such adsorbent [P-CyD/HDI(l/8)]/HDI polymer beads were found to possess good regular shape and high mechanical stability and was prepared by a stepwise crosslinking method.
In this prior art, pre polymer is treated first with HDI and treated in a grounded ball mill which is different from solvent casting method. The crosslinking is performed for cyclodextrin for the removal of effluents, HDI to crosslink agar for biodegradable packaging application.
BOR-SEN CHIOU et. al.: J. Appl. Polym. Sci., 2001, 83: 212-223 discloses the effects of chemical crosslinking on the thermal and dynamic mechanical properties of a polyurethane system were examined. The polyurethanes were prepared from poly(propylene glycol), a diol; trimethylolpropane propoxylate, a triol; and poly(propylene glycol), toluene 2,4-diisocyanate terminated, a diisocyanate monomer. However, polyurethane developed by Chiou et al. are not biodegradable.

Thus, various alternatives have been contrived and efforts are ongoing for finding a suitable biodegradable plastic having improved mechanical properties, biocompatibility, allowing it to be used in varying applications including food/non-food applications, optical applications, drug delivery applications. Accordingly, there continues to be a need for suitable biodegradable plastics exhibiting improved properties over existing plastics for use as alternatives to petroplastics.
OBJECTS OF THE INVENTION:
An object of the present invention is to overcome the drawbacks/disadvantages of the prior art.
Another object of the present invention is to provide a bioplastic material that has good mechanical characteristics with biocompatibility.
Yet another object of the present invention is to provide a process for the preparation of bioplastic which has applications in multiple fields including food/nonfood applications, optical applications, drug delivery applications.
Yet another object of the present invention is to provide improved bioplastic materials having superior mechanical and chemical characteristics with biocompatibility.
Yet another object of the present invention is to provide a process for preparation of bioplastic materials using solvent casting method.
SUMMARY OF THE INVENTION
According to an aspect of the invention, there is provided a process for preparation of a bioplastic material to prepare a film by solvent casting method comprising steps of
i. Preparing a solution of polysaccharides/synthetic polymers dissolved in organic solvents at 80°C;
ii. Preparing a solution of diisocyanates dissolved in organic solvents at 10°C;

iii. Crosslinking the solution of polysaccharides/synthetic polymers with diisocyanates;
iv. Drying the organic solvent by evaporating under hot air oven at 60°C for 36 hours; and
v. Obtaining dried solid crosslinked polysaccharide/polymer diisocyanate films.
According to another aspect of the invention there is provided a bioplastic material comprising polysaccharides/synthetic polymers crosslinked with diisocyanates present in a range of 20% to 40% w/w prepared by present process.
According to another aspect of the invention there is provided an article comprising the bioplastic material.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure la illustrates crosslinking reaction scheme of agar by 4, 4 diphenyl diisocyanate (DDI)
Figure lb illustrates crosslinking reaction scheme of agar by 1, 6 hexamethylene diisocyanate (HDI)
Figure 2a illustrates FTIR spectra of (I) agar (II) 25% w/w (III) 30% w/w 4, 4
diphenyl diisocyanate (DDI) crosslinked agar. Signature bands are represented by short dashed lines
Figure 2b illustrates FTIR spectra of (I) agar (II) 30% w/w/ (III) 35% w/w 1, 6
hexamethylene diisocyanate (HDI) crosslinked agar. Signature bands are represented by short dashed lines
Figure 3a illustrates swelling plot of 4, 4 diphenyl diisocyanate (DDI) crosslinked agar films
Figure 3b illustrates swelling plot of 1, 6 Hexamethylene diisocyanate (HDI) crosslinked agar films

Figure 4a illustrates typical stress-stain plot of (I) 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.
Figure 4b illustrates mechanical properties vs DI concentration (I) 4, 4 diphenyl diisocyanate and (II) 1, 6 hexamethylene diisocyanate (HDI) crosslinked agar
Figure 5a illustrates TGA plot of (I) agar (II) 25% w/w (III) 30% w/w 4, 4 diphenyl diisocyanate (DDI) crosslinked agar
Figure 5b illustrates DTGA plot of (I) agar (II) 25% w/w (III) 30% w/w 4, 4 diphenyl diisocyanate (DDI) crosslinked agar
Figure 6a illustrates TGA plot of (I) agar (II) 30% w/w and (III) 35% w/w 1, 6
hexamethylene diisocyanate (HDI) crosslinked agar
Figure 6b illustrates DTGA plot of (I) agar (II) 30% w/w and (III) 35% w/w 1, 6 hexamethylene diisocyanate (HDI) crosslinked agar
Figure 7 illustrates hemocompatibility of agar and diisocyanates crosslinked agar
Figure 8 illustrates cytocompatibility of agar and diisocyanates crosslinked agar
DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions
DDI-4, 4 Diphenyl Diisocyanate
HDI- 1, 6 Hexamethylene Diisocyanate
Diisocyanates' as used herein means the functional group is isocyanate (N=C=0) at both the ends of aliphatic and aromatic carbon chains.

'Solvent casting method' as used herein means a method where both the polymer and crosslinker are mixed simultaneously and poured into the mould. The mould is then kept in a hot air oven to remove all solvent to obtain dried solid films.
Bioplastics are used in various fields including food/non-food applications, optical applications, and drug delivery applications. They are used as a replacement for petroplastics in packaging applications and as drug-delivery vehicles.
The present invention provides a process for preparation of bioplastic materials having high biocompatibility and high tensile strength. The bioplastic material in accordance with the present invention is prepared by crosslinking polysaccharides/synthetic polymers with hydroxyl groups with diisocyanates to form a film by a solvent casting method.
Previous attempts to crosslink such polysaccharides using di- and tri- carboxylic acids crosslinkers deteriorated its mechanical strength due to chain hydrolysis and use of high temperature (110°C). The present method of crosslinking provides improved water resistance and mechanical strength, reduced water absorption, improved tensile strength and thermal stability for application in biodegradable packaging.
According to an embodiment of the present invention, a solvent casting method for preparing the bioplastics is provided. The 'solvent casting method' is a process for forming polymer samples by transferring polymer solution into a mould and drawing off the solvent to leave a polymer film adhering to the mould. The present technique is simple and fast and cost effective as there is no use of expensive reagents and reduced process steps are included.
The solvent casting method does not need sophisticated equipments and technology as used in other polymer casting techniques like extrusion and injection molding. In less cost, thin films of uniform thickness distribution and maximum optical purity can be prepared using this method. It is a single step synthesis and therefore it takes less time.

In accordance with the invention, the solvent casting method comprises the steps of preparing a polysaccharides/synthetic polymers solution dissolved in organic solvents followed by preparing a solution of diisocyanates dissolved in organic solvents. These two solutions are further crosslinked and the organic solvents are dried and thereby obtained the crosslinked polysaccharide/polymer diisocyanate films. The cross linked films as obtained by the present method is having a thickness of film -50 microns.
According to a preferred embodiment, a process is provided for preparation of a bioplastic material to prepare a film by solvent casting method comprising steps of
i. Preparing a solution of polysaccharides/synthetic polymers dissolved in organic solvents at 80°C;
ii. Preparing a solution of diisocyanates dissolved in organic solvents at 10°C;
iii. Crosslinking the solution of polysaccharides/synthetic polymers with diisocyanates;
iv. Drying the organic solvent by evaporating under hot air oven at 60°C for 36 hours; and
v. Obtaining dried solid crosslinked polysaccharide/polymer diisocyanate films.
The amount of the diisocyanates ranges from 5% to 40% w/w preferably from 20% to
40% w/w.
Suitable polysaccharides/synthetic polymers with hydroxyl groups as used herein can be agar, agarose starch, carrageenan, pullulan, dextran or polyvinyl alcohol.
Suitable solvents as used herein can be Dimethylsulphoxide (DMSO) tetrahydrofuran (THF), acetone, or ethyl acetate.
Agar is extracted from seaweeds. The moderate strength and high water absorption property of agar restricted its applicability as bioplastic material. The inventors in the

present invention have crosslinked agar by diisocyanates to limit the water absorption and improve the mechanical properties. The moderate strength and high water absorption property of agar restricted its applicability as bioplastic material.
The inventors in the present invention have crosslinked agar by diisocyanates to limit 5 the water absorption and improve the mechanical properties.
Agarose is a polysaccharide, mostly extracted from seaweed. It is a linear in nature and consist of repeating disaccharide units of D-galactose and 3, 6-anhydro-L-galactopyranose. It is one of the components of agar other than agaropectin. It is generally used in the separation of large biomolecules like DNA by electrophoresis.
10 Surprisingly the present method does not result in chain scissions of agar and is carried out at room temperature unlike the processes known in the art.
The agar chain scission as disclosed herein means hydrolysis of glycosidic bonds in agar chains into D-galactose and 3, 6-anhydro-L-galactose units. In the present invention, hydrolysis of agar chains is avoided by using diisocyanates as crosslinker. 15 The diisocyanates are not acidic and does not promote cleavage of agar chains.
The diisocyanates according to the invention can be aromatic or aliphatic. Aromatic diisocyanates is 4, 4 Diphenyl Diisocyanate (DDI) and aliphatic diisocyanates is 1, 6 Hexamethylene Diisocyanate (HDI)] for crosslinking agar.
Aromatic diisocyanate, when crosslinked with agar, was more efficient than the 20 aliphatic diisocyanate due to higher reactivity. Diisocyanates (DDI and HDI) and agar are soluble in DMSO. Other diisocyanates [Phenylene Diisocynatae (PDI) and Toluene Diisocyanate (TDI)] are soluble in THF/acetone etc. These solvents, however, do not dissolve agar. It has been confirmed from the FTIR spectra that carbamate crosslink network was formed in the crosslinked agar. The minimum water uptake observed for 25 DDI, and HDI crosslinked agar are 33.6 and 46.3% respectively in comparison to agar (206%). The DDI crosslinked agar has a maximum strength of 45.3 MPa which is higher than HDI crosslinked agar (30.6 MPa) and agar (31.7 MPa).
1 1

The mechanical properties in terms of tensile strength and elongation at break for agar, for DDI and HDI crosslinked agar are summarized in Table 1. Figure 4a represents typical stress-strain plot of agar, DDI and HDI crosslinked agar samples. Crosslinking of agar leads to the formation of a large number of crosslinks that restricts the mobility 5 of polymer chains and hence results in improved strength of the polymer with a decrease in elongation at break. The mechanical properties also depend upon the geometry of the crosslinker, which is observed in the case of two diisocyanates used in this study.
The DDI crosslinked agar show 37% higher tensile strength and 8.9% less elongation at 10 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.
15 The tensile strength of crosslinked agar decreases at higher concentration of diisocyanates. There could be two opposing factors playing: an increase in crosslinking density with higher crosslinker concentration results in improvement of strength. Application of a large amount of crosslinker, on the other hand, leads to unused diisocyanates in agar matrix, which upon washing may leave behind micropores causing
20 a reduction in strength. The swelling data also confirms that degree of crosslinking does not change at extreme diisocyanate concentrations.
According to another embodiment there is provided a bioplastic material comprising polysaccharides/synthetic polymers crosslinked with diisocyanates present in a range of 20% to 40% w/w prepared by solvent casting method. The aromatic diisocyanate is 4, 4 25 diphenyl diisocyanate (DDI) and the aliphatic diisocyanate is hexamethylene diisocyanate (HDI).
The bioplastic material as obtained in the present invention provides an improved tensile strength ranging from 3625-5760 psi.
1 2

In accordance with the invention, an article comprising the bioplastic material is provided. The bioplastic material can be useful in many applications e.g. as a drug delivery vehicle.
Advantages of present invention over prior art

Prior art Present Invention
US 3860573A ‘Method for crosslinking agarose or agar’
A method for crosslinking agarose or agar, wherein the agarose or agar is suspended and then reacted with a bifunctional compound with a functional group (-COCl, -SO2Cl, and -N=C=S). The crosslinked agarose or agar is finally filtered and washed.
US4591640 ‘Method for crosslinking of agar products’
The invention relates to a method for crosslinking of agar products in an alkaline surrounding (pH>11) using divinylsulfone.
US5371208 ‘Preparation of crosslinked linear polysaccharide polymers as gels for electrophoresis’
A bed of transparent, water insoluble, charge-free crosslinked gel is prepared. (Crosslinking agents are bis-epoxides, halo-epoxides, bis-haloalkanes, bis-halo-alcohols, alkanediol-bis-alkyl sulfonates, alkanediol-bis-aryl sulfonates or divinylsulfone. The liner polysaccharide maybe mixed with a synthetic polymer such as polyvinyl alcohol. Crosslinker chosen is similar as reported in this reference i.e. bifunctional but the functional group is isocyanate (N=C=O) at both the ends of aliphatic and aromatic carbon chains.
Effect of crosslinking
The effect of crosslinking has been studied on water absorption, thermal and mechanical properties of agar.
Use
The crosslinked material has use in bioplastic packaging material and biomedical applications.
1 3

Use
For electrophoresis
US4665164 ‘Polysaccharide crosslinked separation material and its preparation’
A separation material of crosslinked agarose in which the agarose is crosslinked using at least one bi- or polyfunctional crosslinking agent, which gives a chain of 6 to 12 atoms between two binding points, on one hand and using at least one bifunctional crosslinking agent, which gives a chain of 2 to 5 atoms between two binding points on the other.
The separation material is prepared by crosslinking agarose in two steps with (a) a bi- or polyfunctional crosslinking agent, which gives a chain of 6 to 12 atoms between two binding points and (b) a bifunctional crosslinking agent, which gives a chain of 2 to 5 atoms between two binding points, preferably in the order mentioned. Crosslinker chosen is similar as reported in this reference i.e. bifunctional but the functional group is isocyanate (N=C=O) at both the ends of aliphatic and aromatic carbon chains
The effect of crosslinking has been studied on water absorption, thermal and mechanical properties of agar.
US4992220 ‘Method for producing biodegradable packaging material’
A mixture of boiling water and a gelling agent, such as the polysaccharide agar, are mixed and poured into a mold and allowed to cool and gel. The gelled mixture is then removed from the mold and frozen, after which it is placed in a freeze drier where it is completely sublimated to remove therefrom all moisture. The resultant freeze-dried material is extremely light in weight, is biodegradable, and exhibits favorable fire Agar based bioplastic is developed by crosslinking of agar with reduced water absorption and improved thermal and mechanical properties.
Crosslinked Agar film is prepared by solvent casting method using DMSO (dimethylsulfoxide)
1 4

retardant properties.
906/DEL/2015 ‘A process for preparation of bioplastics’
Demonstrated crosslinking of agarose using citric acid. Swelling resistance and strength first improved but the properties deteriorated at higher crosslinker concentration since crosslinker also degraded agarose by hydrolyzing agarose chains. Crosslinkers are diisocyanates that does not cause chain scission of agar.
The examples are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. Efforts have been make to ensure accuracy with respect to numbers used, but some experimental errors and deviations 5 should be accounted for.
Example 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
10 diisocyanate (HDI) were procured from Sigma Aldrich, India. Dimethylsulphoxide (DMSO) and tetrahydrofuran (THF) were purchased from Merck, Germany. Polypropylene (PP) petridishes were purchased from Tarsons Product Pvt Ltd. For phosphate buffered saline (PBS) solution, potassium chloride, sodium chloride, potassium dihydrogen orthophos- phate, disodium hydrogen orthophosphate were
15 purchased from Thermo Fisher Scientific (Mumbai), India. 3-(4,5-Dimethylthiazol-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,
1 5

Thermo Fisher, India. Ethanol was purchased from Merck specialties Private Limited (Mumbai).
Preparation of agar and diisocyanates crosslinked films
Agar 1 g was dissolved in 40 mL DMSO at 80°C. The solution was then cooled down 5 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 of crosslinker (DDI or HDI) was dissolved in 10 mL of DMSO at 10°C. The agar solution (40 mL) was transferred into crosslinker solution (10 mL) and was mixed vigorously using a magnetic stirrer for 10-15 minutes. The solution was then
10 poured into polypropylene petridish and allowed to react for 12 hours at room temperature. The petridish was kept in a hot air oven 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 remove unreacted diisocyanate. Washing was performed using a mechanical shaker for 12 hours and then
15 the films were dried at 60°C to remove THF.
Characterization of films
Swelling study was performed to measure the amount of absorbed water according to the ISO standard (ISO 62:2008, Plastics - Determination of water absorption). Agar films were cut into three square pieces (2 cm x 2 cm), weighed and immersed in DI 20 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 weighed.
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 25 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 D882-12 standard was used for testing
1 6

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 moisture in them. Strips were stretched at a rate of 10 mm/minute at 50% relative humidity (RH) as 5 per the above standard.
Thermogravimetric analysis (TGA) of neat and crosslinked agar films was performed using Perkin Elmer STA 8000 in the range 30°C-600°C at a heating rate of 10°C per minute under nitrogen gas.
Hemocompatibility assay: For the Hemocompatibility assay, samples were washed 10 thrice with 1×PBS. The assay was performed as elsewhere (Fischer et al., Biomaterials 24(7) (2003) 1121-1131). The hemolysis percentage of the samples is defined as:

where OD is the optical density of the samples measured at 540 nm.
Biocompatibility assay: For biocompatibility assay, NIH 3T3 (NCCS) cells were 15 cultured in the high-glucose DMEM medium containing 10% FBS and penicillin-streptomycin (10000 U/mL) and incubated in a humidified environment with 5% CO2 at 37°C. 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 then later incubated for 24 h in incomplete DMEM in 48 well 20 micro-titer plates.
The cells were harvested after 70% confluence using trypsin solution. 5×104 cells/well in complete media were seeded into 48 well plate and were incubated further for 7 days. 2D cell culture on tissue culture plate (TCP) well was used as a control on which an equal number of cells were seeded. At predefined time intervals, DMEM media was 25 removed from the well and replenished with 200 µL MTT solutions (0.5 mg/mL in incomplete DMEM medium). After 4 h incubation, media was replaced by 500 µL DMSO and incubated for 10 more minutes under the same condition. 100 µL solutions
1 7

from each well were transferred to a 96-well plate and the color change was measured at a wavelength of 570 nm by using an ELISA plate reader.
Result and Discussion
Crosslinking reaction scheme
5 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 10 this manuscript we have considered intermolecular crosslinking.
Figure 2a, and 2b are 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 (–NH–C=O–) bands in figure 2a, and 2b.
15 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 et al., Polymer 41(26) (2000) 9265-9272). The peak position of the characteristic bands is same for DDI, and HDI crosslinked agar samples.
Swelling study - water uptake
20 A large number of hydroxyl groups present on agar attracts water molecules. The incoming water occupies space between agar chains by displacing them and hence and cause swelling. The absorbed water also reduces the bonding between agar chains. Here, swelling of agar is reduced by crosslinking its chains by diisocyanates. Crosslinking not only reduces the number of hydroxyl groups but also restricts the
25 mobility of agar chains by chemically crosslinking them. Therefore, in crosslinked agar, there is very less number of water molecules absorbed in the interspaces of polymer chains and they are unable to cause mobility in the polymer chains. The swelling percentage in diisocyanate crosslinked agar samples is calculated by using equation (1)
1 8

Swelling (%) = WS WDx100 (1)
D
where WS is the weight of swollen samples and WD is the weight of dry samples.
Swelling percentage decreases with increasing diisocyanate (crosslinker) concentration in agar (figure 3). It indicates a reduction in the number of hydroxyl groups and 5 formation of a large number of carbamate crosslinks in agar that restrict the mobility of polymer chains, hence a reduction in the 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 and HDI crosslinked agar could be due to the difference in reactivity.
10 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 is get delocalized into R, which makes aromatic diisocyanate (DDI) more reactive in comparison to aliphatic diisocyanate (HDI) (Sharmin et al,
15 Polyurethane: An Introduction, INTECH Open Access Publisher 2012), hence it reacts with larger number of hydroxyl groups of agar.
The swelling plots (figure 3a and 3b) justify the above explanations. In figure 3a, it is observed that swelling percentage decreases successively with increasing DDI concentration and after critical concentration of 25% w/w DDI, hardly any difference in
20 swelling percentage is observed. It indicates the saturation in swelling resistance. Similar to the trend of figure 3a, the continuous decrease in the swelling percentage with increasing HDI concentration can be observed in the figure 3b. However, the critical concentration for DDI is 25% w/w and for HDI is 35% w/w. Even at lower concentrations, DDI crosslinked agar restricts swelling more effectively in comparison
25 to HDI. It is due to the reactivity difference between the crosslinkers as explained earlier.
Table 1. Swelling percentage and mechanical properties of agar, DDI and HDI
crosslinked agar samples. 1 9

Agar films Swelling % Tensile Strength (MPa) Elongation at break (%)
Agar 206 (6.6)* 31.7 (1.3) 13.8 (1.4)
DDI crosslinked Agar
5% w/w 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)
15% w/w 72.7 (4) 37.7 (1.6) 5 (0.5)
20% w/w 55.6 (4.8) 43.9 (0.9) 6.2 (0.9)
25% w/w 34.6 (5) 43.5 (3.2) 4.9 (0.5)
30% w/w 33.2 (5.5) 45.3 (2.3) 3.8 (0.4)
35% w/w 34.4 (2) 32.6 (1.8) 2.6 (0.1)
40% w/w 33.6 (6.1) 34.4 (1.8) 3.4 (0.1)
HDI crosslinked Agar
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)
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)
*Standard error of the mean values
Tensile Testing
5 The mechanical properties in term of tensile strength and elongation at break for agar, DDI and HDI crosslinked agar are summarized in Table 1. Figure 4a represents a stress-strain diagram of agar and DDI and HDI crosslinked agar samples. The strength of aromatic DDI crosslinked agar is greater than the HDI crosslinked agar (figure 4b). It is
2 0

due to effective crosslinking of agar by aromatic diisocyanate in comparison to aliphatic diisocyanate.
Thermal analysis
Crosslinking of agar by diisocyanates improve its thermal stability at higher 5 temperatures (figure 5). The TGA graphs of agar and crosslinked agar are analyzed in three regions. The first 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 samples, agar, DDI, and HDI crosslinked agar show similar weight loss in this region.
10 The second stage, from 200-500ºC, corresponds to chain scission and degradation of agar 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% weight in this temperature range while agar has retained only 40% weight (figure 5a).
15 HDI crosslinked agar samples also demonstrate thermal resistance (figure 6a), which is not as 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 provides higher thermal stability than HDI. Moreover, at high temperatures, aromatic diisocyanate containing polyurethane are thermally more stable than the
20 aliphatic diisocyanates (Chattopadhyay et al., Journal of Applied Polymer Science 95(6) (2005) 1509-1518).
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 5b) of DDI 25 crosslinked agar shows no shift in the decomposition temperature in comparison to agar (250°C). However, the rate of decomposition is slow as DDI crosslinked agar becomes thermal resistant due to crosslinking. Similarly, HDI crosslinked agar also does not show a shift in the decomposition temperature with a slow rate of degradation in comparison to agar (figure 6b). Moreover, HDI crosslinked agar showed one additional
2 1

peak of decomposition at 450°C. It could be due to decomposition of the modified crosslinked network. In DDI crosslinked agar, this peak is absent as aromatic diisocyanate crosslinked network has higher thermal resistance in comparison to HDI crosslinked agar.
Hemocompatibility test
The hemolysis % graph (Fig. 7) indicates that the agar- diisocyanates samples show less than 5% hemolysis, hence the samples could be considered as hemocompatible. Materials showing hemolysis less than 5% are considered as hemocompatible (Autian et al., Polymers in Medicine and Surgery, Springer US, Boston, MA, 1975, pp. 181-203). Therefore, the crosslinked films could also be used for bio-applications.
Biocompatibility test
MTT assay results (Fig. 8) showed that all the agar-based diisocyanate samples are suitable for fibroblast (NIH 3T3) cell growth. The cell viability numbers varying from 65% to 85% show that the crosslinked agar films are slightly cytotoxic in nature. MTT method is widely used for the preliminary biocompatibility evaluation of the biomaterials (Mansur et al., Materials Science and Engineering: C 29(5) (2009) 1574-1583). The biocompatibility assay is performed on the films taking 2D TCP is taken as positive control. The reduction in cell viability could be due to surface properties like wettability and some degradation products (Barrioni et al., Materials Science and Engineering: C 52(Supplement C) (2015) 22-30).
It is important to note that the obtained mechanical properties of crosslinked agar depend on the geometry of crosslinker. DDI crosslinking can be used to develop high strength crosslinked material whereas HDI crosslinking would yield a crosslinked material with higher elongation. Figure 7 and 8 shows the hemocompatibility and cytocompatibility of the processed agar samples which can be used in the biomedical applications.

A process for preparation of a bioplastic material to prepare a film by solvent casting method comprising steps of
i. Preparing a solution of polysaccharides/synthetic polymers dissolved in organic solvents at 80°C;
ii. Preparing a solution of diisocyanates dissolved in organic solvents at 10°C;
iii. Crosslinking the solution of polysaccharides/synthetic polymers with diisocyanates;
iv. Drying the organic solvent by evaporating under hot air oven at 60°C for 36 hours; and
v. Obtaining dried solid crosslinked polysaccharide/polymer diisocyanate films.
The process as claimed in claim 1, wherein said polysaccharides/synthetic polymers with hydroxyl groups is selected from agar, agarose, starch, carrageenan, pullulan, dextran or polyvinyl alcohol.
The process as claimed in claim 1, wherein said diisocyanates is capable of preventing chain scission of agar.
The process as claimed in claim 1, wherein the amount of the diisocyanates ranges from 5% to 40% w/w.
The process as claimed in claim 4, wherein the amount of the diisocyanates ranges from 20% to 40% w/w
The process as claimed in claim 1, wherein said the film is having a thickness of -50 micons.

A bioplastic material comprising polysaccharides/synthetic polymers crosslinked with diisocyanates present in a range of 20% to 40% w/w prepared by the process of claim 1.
The bioplastic material as claimed in claim 7, wherein said diisocyanates are aromatic or aliphatic.
The bioplastic material as claimed in claim 8, wherein said diisocyanates are aromatic.
'. The bioplastic material as claimed in claim 7, wherein said aromatic diisocyanate is 4, 4 diphenyl diisocyanate (DDI).
. The bioplastic material as claimed in claim 1, wherein said aliphatic diisocyanate is hexamethylene diisocyanate (HDI).
. The bioplastic material as claimed in claim 7, wherein said bioplastic is having a tensile strength ranging from 3625-5760 psi.
. An article comprising the bioplastic material as claimed in claims 7-12.
. An article as claimed in claim 13, wherein said article is a drug delivery vehicle.