WO2016156930A1

WO2016156930A1 - A process for preparation of bioplastics

Info

Publication number: WO2016156930A1
Application number: PCT/IB2015/053216
Authority: WO
Inventors: Ankur AWADHIYA; Vivek Verma
Original assignee: Indian Institute Of Technology - Kanpur
Priority date: 2015-03-31
Filing date: 2015-05-02
Publication date: 2016-10-06

Abstract

The present invention is in the field of bio-plastics. The invention particularly provides a process for preparation of agarose based bioplastic and an agarose based bioplastic thereof. The bio-plastic has good biocompatibility and mechanical strength. The bioplastic has uses in multiple fields including food/non-food applications, optical applications, drug delivery applications.

Description

A PROCESS FOR PREPARATION OF BIOPLASTICS

FIELD OF THE INVENTION

The present invention relates to a process for preparation of bioplastics. More particularly, the present invention relates toa process for preparation of agarose based bioplastic materials and applications thereof.

BACKGROUND OF THE INVENTON

The fabrication of bioplastics began in the 1970's with the use of starch in polymer blends and as a filler material in petro-plastics. This interest in the use of renewable bioplastics has been linked to the emerging environmental movement. While on the one hand the quality of life improved significantly with rising affluence and new products; on the other hand, people started to be wary of long-term effects of the new chemicals in those products on their lives and those of their children.

Bioplasticsare suitable alternatives as they are expected to mimic the competent properties of petro-plastics that they seek to replace. Hence, their design is commonly based on similar constituent molecules, namely,longchain polymers. Many biological macromolecules are long chain polymers.However, it is carbohydrates, especially starch, that occupy the dominant position as raw materials for bioplastics. They are abundantly and cheaply available from food and non-food crops, are quick to biodegrade, and are easy to process.

Starch, in particular- destractured or destructurized starch, is however generally hard and brittle in the dried state or even at low water contents below 10%, and cannot be moulded at high temperatures typically employed in thermoforming or moulding shaped articles, sheets or films.

One such plastic is called thermoplastic-like starch or TPS. Starch is not a real thermoplastic; however, in the presence of high temperature, shearing and plasticiser (water, sorbitol, glycerol, etc.), it melts and turns into a fluid. However, a major problem with starch based bioplastics is their mechanical strength. Additives have been used to improve the mouldabilityby reducing the melting temperature of starch below its decomposition temperature. However, resulting products arebe unstable and properties diminish in time due to migration or even loss of plasticizers during storage and use. Furthermore, products are highly water and humidity sensitive. US 7,148,272 discloses a biodegradable material based on a dispersive blend, e.g. a homogeneous mixture of polymer and cereal flour. The polymer is selected for its mechanical properties and its ability to be extruded. In the process the cereal flour is first subjected to a controlled drying in order to eliminate the moisture so as to avoid excessive degassing during subsequent moulding steps. As demonstrated in figure 1, it is only after drying that the dried flour granules and plastics, such as non-biodegradable polypropylene, are moulded together by (dry blend) dual injection, thus yielding a dispersive blend of inadequately compatible components having properties which are considered inferior for the applications in the field of the invention; for instance, the mechanical properties, such as elongation, will decrease and more brittle products will be obtained. US 20130186303 disclosesbioplastics based on an oligosaccharide, a plasticizer, and an additive. Such bioplastics display advantageous attributes including tensile strength that can be tailored for particular uses. However, the maximum tensile strength for this material is too low, only 7.3 MPa under most optimum conditions, which makes it unsuitable for multiple applications. US 20080090939 relates to soy protein-based polymeric compositions and, more particularly, to biodegradable polymeric compositions containing soy protein in combination with polysaccharide strengthening agents. However, in view of high protein content, this material becomes inappropriate for use as a biomaterial, due to allergic responses to proteins in a majority of population. US6235816relates to a method to manufacture a biodegradable thermoplastic mixture comprised (i) combining pre-dried starch and a plasticizer agent to form a molten thermoplastic starch with water content below 1%; (ii) combining at least one polymer selected from the group comprising: aromatic polyesters, polyester copolymers of aromatic and aliphatic blocks, polyester amides, polyethylene oxide polymers, polyglycols, and polyester urethanes with the molten thermoplastic starch and an aliphatic polyester (PLA, PCL, polyhydroxybutyric acid or copolymer of polyhydroxybutyric and hydroxyvaleric acid), where the thermoplastic starch comprises between 10 and 95 weight% of the starch/polymer mixture and the stage is carried out at one or more temperatures in the range from 120 to 260°C, preferably between 140 and 160°C; (iii) Solidifying the mixture in water and permitting the mixture to reabsorb water to a content in the range of 1 to 6 weight%. However, the use of non-biocompatible materials in production and processing makes it inappropriate for biomedical applications such as drug delivery.

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 overexisting 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 an agarose based bioplastic material that has good mechanical characteristics with biocompatibility.

Yet another object of the present invention is to provide a process for thepreparation of agarose based bioplastic which have applications in multiple fields including food/non food applications, optical applications, drug delivery applications. Yet another object of the present invention is to provide modified agarose based bioplastic materials that havesuperior mechanical and chemical characteristics with biocompatibility.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a process for preparation of a bioplastic material, comprising steps of a) mixing and heating of agarose with water and a plasticizer to boiling temperature of 90°C to 100°C; b) cooling of the solution from step (a) to a temperature of about 60 °C; and c) casting solution from step (b) into sheets wherein the amount of the plasticizer ranges from 1 to 50% w/w.

In another aspect, the present invention provides agarose based bioplastic materials having good mechanical and chemical characteristics with biocompatibility.

In yet another aspect, the present invention provides agarose based bioplastic materials having applications in multiple fields including food/non food applications, optical applications, drug delivery applications.

In a further aspect, the invention provides modified agarose based bioplastic materials having superior mechanical and chemical characteristics with biocompatibility.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 illustrates dimensions of specimen of bioplastic used for tensile testing.

Figures 2a, 2b and 2c illustrate representative images of agarose based bioplastic films.

Figure 3a, 3b, 3c and 3d illustrate tensile properties of bioplastic films which is a function of the concentration of plasticizer.

Figure 4 illustrates representative IR spectra of agarose based bioplastic films without glycerol (continuous line; lower curve) and with 20% glycerol (dashed line; upper curve). Figure 5 illustrates swelling curves of agarose - 20% glycerol bioplastic films at 4°C, 25°C and 37°C as a function of time.

Figure 6(a to f) illustrate degradation studies of polyethylene sheet (a, d), agarose based bioplastic film (b, e) and filter paper (c, f) which were kept under moist conditions.

Figure 7 illustrates water contact angle measurement of agarose based bioplastic surface

Figure 8 illustrates adsorption of bovine serum albumin on agarose based bioplastic and tissue-culture polystyrene (TCPS, control) surfaces.

Figure 9 illustrates direct and indirect contact cell culture assays for assessment of the cytocompatibility of agarose based bioplastic.

Figure 10 illustrates haemocompatibility assay of agarose based bioplastic sample vis-a-vis phosphate-buffered saline pH 7.4 (negative control) and 2% Triton-X 100 (positive control). Figure 11 illustrates representative curve for the rate of release of ampicillin, a model drug, from agarose based bioplastic films.

Figure 12(a to f): illustrates study of potencies of drugs incorporated in agarose based bioplastic, as determined through agar diffusion assay.

Figure 13 illustrates the variation in the strength of composites as a function of amount of bacterial cellulose reinforcement. The strength of bioplastic increases with increasing cellulose before decreasing again. Maximum strength observed was for 20% w/w of bacterial cellulose.

Figure 14 illustrates Swelling curves of composite bioplastic films with 20% bacterial cellulose at 4°C, 25°C and 37°C as a function of time. The amount of water absorbed increases with temperature and time till a plateau is reached in around 120 minutes.

Figure 15a and 15b illustrate the fourier transform infrared spectroscopy of crosslinked and control films. The spectrograms of the crosslinked films were similar to the control films, with one region of major difference, indicated by an arrow.

Figure 16 illustrates the crosslinking performed over for larger periods of time the increased amount of crosslinking increased the strength of the bioplastic. Figure 17 illustrates the swelling curves of crosslinked bioplastic films optimized for strength at 4°C, 25°C and 37°C as a function of time.

Figure 18 illustrates the effect of crosslinking on degradation behavior. While control and crosslinked films were completely degraded in 5 hours (black bars), the amount of degradation of control films at 30 minutes was less than the amount of degradation of crosslinked films at 30 minutes (grey bars).

Figure 19 illustrates thermograms of crosslinked (black) films and non-cross linked control (grey) films. The crosslinked films absorbed less quantity of water, had slower rate of thermal degradation and generated a higher percentage of ash content.

Figure 20a to 20c illustrate characterisation of films through SEM and EDS. While control films (a) had a smooth surface topography, films loaded with silver nanoparticles (b) had punctate structures. These punctate structures were sub-micron in size. EDS characterisation showed a distinct peak for silver (c), confirming the punctate to be silver nanoparticles.

Figure 21a and 21b illustrate an assay for anti-bacterial properties of silver nanoparticle incorporated bioplastic films. Agar diffusion assay revealed that while the control films without silver nanoparticles did not create a zone of inhibition (a), the films loaded with silver nanoparticles had a distinct zone of inhibition (b). The dimensions of the films in each case are 1 cm x 1 cm. DETAILED DESCRIPTION OF THE INVENTION

The following description is intended to describe working of the present invention, and not intended to limit the scope of the invention. Other embodiments may also be covered under the scope of the invention. Bioplasticsare 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 bioplasticmaterials composed of agarose that have high biocompatibility and high tensile strengthcomprising steps of;

1. Mixing and heating of agarose with water and plasticizer ranges from 1% to 50%;

2. Cooling of agarose solution: and

3. Casting said resulting agarose solution into sheets.

Surprisingly the present process does not require addition of strong acids and other non- biocompatible materials unlike processes known in the art.

Further, the raw material being agarose is commercially available in abundance. Such agar is purified to have only straight linear chains. It is converted to bioplastic films using the gelatinisation route involving steps of mixing and heating agarose powder with water and a plasticiser and casting into sheets. Glycerol is used as a plasticizer in the present invention. Upon drying these sheets gave rise to thin, transparent and strong films. As illustrated in figure 2a, the cast films of agarose and glycerol as prepared by the present process are transparent. Their thickness varies from 130μπι to 170μπι depending on the amount of glycerol, which is used in a range between 1% and 50% (v/w), relative to the weight to agarose. These biocompatible and biodegradable bioplastic materials (agarose based bioplastics) have myriad applications and uses, which range from food/non food applications, optical applications to drug delivery applications.

Films with lower concentrations of the glycerol are relatively inflexible; however, with increasing concentrations of glycerol, the films are softer and succulent. Inflexible films are good for applications that require structural rigidity; examples include packaging boxes. They, however, are not ideal for wound drug delivery applications, for they being relatively hard may scratch or cut the skin. Moreover, since many body parts are not flat, the dressings should be flexible enough to wrap around body parts. Soft, succulent films arefound to be better for such applications, since they arequite flexible and canbe bent or wrapped as illustrated in figures 2(b) and (c). In addition to being soft and flexible, the films for drug delivery applications are also strong enough to resist wear and tear during application and bodily movements. Figure 2 illustrates agarose based bioplastic films. The films are visually transparent (a), strong and flexible (b, c). In figure (a), the film was put over a logo which shows the transparency quality of the film. Therefore, the amount of glycerol to be added depends on the end use of such plastics and can be modulated accordingly.

In an embodiment of present invention, the agarose based bioplastic may serve as a good drug delivery vehicle for topical applications because of its characteristics. It is made from natural materials, agarose and glycerol, which are known biocompatible materials and are well tolerated by the human body. Agarose has long been used in the culture of cells, while glycerol forms the base of many ointments.

In an embodiment of the present invention, bacterial cellulose is added to theagarose basedbioplastic of the present invention in a concentration ranging from 10% to 50%. As known to those skilled in the art, addition of bacterial cellulose causes reinforcement on the tensile strength and swelling characteristics.In the present invention, addition of bacterial cellulose exhibited remarkable enhancement of 140% in the tensile strength of the agarose based bioplastics.

In another embodiment of the present invention, the bioplastic of the present invention is chemically crosslinked with citric acid. The agarose based bioplastic is prepared according to the present process. Varying concentrations of citric acid and sodium hypophosphite (50% w/w of citric acid) are added to the molten agarose based bioplastic. The solution is swirled manually again, before casting into films. The bioplastic films are thoroughly dried. In yet another embodiment silver nanoparticles are incorporated in the agarose based bioplastic of the present invention by methods known to those skilled in the art. Silver nanoparticles are incorporated through serial imbibition of the bioplastic in silver nitrate and sodium borohydride solutions improve the antibacterial properties of the agarose based bioplastic prepared by present process.

Silver-incorporated agarose-based bioplastic exhibited improved antibacterial properties. The anti-bacterial properties of the films were tested through agar-diffusion assay. These films with anti-bacterial properties can be cheap, biodegradable and ecologically sustainable alternatives to petroleum-derived plastics. In packaging applications, the anti-bacterial activity provided by silver nanoparticles is useful as it willenhancethe shelf-life of products, while in hospital and house -hold settings, these help prevent transmission of pathogens.

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 should be accounted for.

Example 1

Fabrication of agarose based bioplastic In 250 mL Erlenmeyer flasks, two grams of agarose powder was added to 30mL deionised water. Different amounts of glycerol were added to give a final glycerol concentration varying from 1% to 50% (v/w) with respect to agarose. Deionised water was further added to make the final volume up to 50 mL. The mixture was brought to a boil in a microwave oven set at 800 W for

4 minutes, which turned agarose into a viscous solution. The solution was allowed to cool to around 60°C and then manually swirled before casting into films. To make bioplastic films, twenty milliliters of the solution was poured into 90 mm petri plates, cooled and dried at 25 °C for 72 hours at 50% relative humidity.

Fourier transform infrared spectroscopy analysis indicated that no new chemical moieties were formed during processing.

Testing for mechanical properties

The thicknesses of the films were measured using Digimatic micrometer (Mitutoyo Corporation, Japan) and were found to lie between 130 and 170 μπι. Therefore, ASTM D 1708-59 T standard was followed to measure the tensile properties of the samples. Briefly, the samples were cut into dog-bone shaped specimens [as shown in Figure 1] using Epilog Laser C0₂ laser engraving and cutting machine. They were incubated at 25 degree celisus and 50% relative humidity for 72 hours, and then tested on Instron 1195 tensile testing machine with an initial grip separation of 0.9 inches and crosshead speed of 1 mm / min as per the above ASTM standard. Multiple specimens were tested for each sample, and the average ultimate tensile strength was recorded.

Mechanical properties of bioplastic films The ultimate tensile strength of films reduced from around 66 MPa to a little above 10 MPa, with increasing concentration of glycerol from 1 to 50% (v/w, relative to the amount of agarose) [Figure 3]. The elongation at break, on the other hand, increased from around 10% to 40% with higher concentrations of glycerol. For further studies, films with 20% glycerol were chosen

Fourier transform infrared spectroscopy

Samples of the films were dried for five days in a vacuum desiccator lined with anhydrous silica gel, before being ground into a powder. FTIR spectra of the powders were taken using Perkin Elmer Spectrum Two spectrometer in the wavenumber range from 400 cm to 4000 cm^-1.

The FTIR spectrum of pure agarose film is shown in figure 4. The large trough seen at 3500- 3200 cm^"1 is indicative of the large number of hydroxyl groups present both in agarose as well as in glycerol. Other troughs indicative of 3, 6 - anhydrogalactose (930 cm^"1 and 1070 cm^"1), C-H stretching (3000-2850 cm^"1) and other functional groups were also seen. It was observed that the spectra of films without glycerol and those with 20% glycerol were similar with no obvious difference in troughs [as evident from figure 4]. While this was expected since the bonds present in glycerol (C-H, C-O, O-H, C-C) are also present in agarose, the emergence of no new troughs also indicated that no new covalent bonds were formed by the presence of glycerol. The similarity in the curves shows that no new covalent bonds are formed in the film with glycerol, and that the constituents exist as a physical mixture, without reacting chemically.

Swelling behaviour studies

The swelling studies of present bioplastic material in water were performed for up to 135 minutes at three temperatures: 4°C, 25°C, and 37°C, to mimic the conditions of cold weather, normal weather and body temperature, respectively. The percentage of swelling (Sw), defined by the equation:

weight after swelling— initial eisht

= - : — X 100

InitiGiweigiit

Was plotted as a function of time elapsed as shown in Figure 5, which shows that the amount of water absorbed increases with time and temperature till a plateau is reached. Water baths containing deionised water for each of the above temperatures were set. Small strips of bioplastic films (size 1 cm x 3 cm) were cut and weighed. A strip of the bioplastic film was immersed in each water bath. After every 15 minutes, the strips were taken out, wiped with filter paper, weighed and then put back into their respective water baths. The measurements were continued for a little over two hours, by when the successive readings had equalised.

It was observed that the rate of absorption of water was highest in the beginning for all temperatures, and then gradually decreased with time, as the films became saturated. The saturation was complete in 120 minutes. The amount of water absorbed by the films increased with temperature. At 37°C, the amount of water absorbed was more than 700% of the weight of the film. On the other hand, absorption of water at 4°C was around 500% of the original weight of the film. Further significant swelling occurred within fifteen minutes. The large amount and fast rate of swelling indicates that the chains are relatively unhindered in their movement thus being highly suitable for applications that demand a quick release of drugs, such as drug delivery to wounds.

Biodegradation studies Degradation studies were performed under aerobic conditions. Agarose based bioplastic materials prepared by the present process were swollen with water and kept in a petridish, exposed to ambient conditions in the laboratory. A sheet of biodegradable filter paper (Whatman filter paper no. 1) was used as a positive control while a sheet of nonbiodegradable polyethylene served as a negative control. Both of these were cut to same size as the bioplastic film, and kept moist under similar conditions. All the samples were sterilised before use by soaking in 70% ethanol for five minutes and drying. The films were imaged every week for any changes. At the end of the experiment, the films were removed, dried in vacuum desiccators for three days, and then imaged on a scanning electron microscope (Zeiss EVO50) after gold coating through sputtering. Figure 6 illustrates the results of degradation studies. The aerobic microbial degradation as a proxy for the assessment of degradability of the bioplastic. To assess degradability, bioplastic films, and films of polyethylene (negative control) and filter paper (positive control) were kept moist in petri plates and exposed to air in ambient laboratory conditions. The specimens were observed for several weeks for any microbial growth or other changes. It was observed that while there were no discernible changes in the non-biodegradable polyethylene film [Figures 6(a) and (d)] ; the biodegradable filter paper and bioplastic films had perceptible changes [Figures 6(b), (c), (e) and (f)]. There was a heavy growth of microbes on the bioplastic film after three weeks. At many places where there was a dense proliferation of microbes, the film had a visually dented appearance with holes, which were a telltale sign of the degenerating film. Similar changes were also observed for the filter paper, though on a more modest scale. The microbial colonies observed in the case of bioplastic films and filter paper were dark green in colour and had a mushy and capillaceous appearance, suggestive of fungal growth. Scanning electron micrographs revealed the presence of hyphae, confirming the presence of moulds. The images show the appearance of films on Day 0 (a, b, c) and after three weeks (d, e, f). While there was no discernible microbial growth on polyethylene sheet, the bioplastic film and filter paper showed the presence of dark green colonies of moulds. Scanning electron microscopy of the microbial colonies revealed the existence of fungal hyphae (g), confirming the presence of moulds. The diameter of petri dishes (a-f) is 90 mm.

This study validates the biodegradability of the agarose based bioplastic films. The biodegradability of agarose based bioplastic was even more so than filter paper, which was used as a positive control in the experiments.

Example 3

Fabrication of agarose based bioplastic and loading of drugs

In 250 mL Erlenmeyer flasks, two grams agarose powder and 0.4 mL glycerol were added to 30 mL deionised. Deionised water was further added to make the final volume up to 50 mL. The mixture was brought to boil in a microwave oven set at 800 W for four minutes, which turned the mixture into a viscous solution. The solution was allowed to cool to around 60°C and then manually swirled before casting into petri plates. To make the films, twenty milliliters of the solution was poured into 90 mm petri plates, cooled and dried at 25°C for 72 hours at 50% relative humidity. To incorporate drugs (antibiotics and antiseptics) into the bioplastic films, the drugs were added into the agarose-glycerol solution just before casting. The drugs were not added until immediately prior to casting to prevent any degradation due to the high temperature processing step. The addition was accompanied by continuous swirling to facilitate thorough mixing of the drug with the bioplastic.

Example 4

Biocompatibility of bioplastic films as determined by contact angle measurements

The contact angle measurements were performed using OCA 35 video based automatic contact angle measuring device procured from DataPhysics Instruments GmbH. Briefly, 20μ1 drop of deionised water was put on the surface of bioplastic film, and was imaged. A horizontal line was defined in the software module, which then measured the angle made by the water drop with the horizontal line. The software calculated two readings for each sample, left and right. The average of multiple readings obtained by repeating these steps on several surfaces of the bioplastic was recorded as the water contact angle. The results are shown in figure 7 which shows that when a drop of water was placed over the bioplastic surface, it spreads. The contact angle made by the interaction of water with the bioplastic surface was measured using software. The low value of contact angle indicated that the material is hydrophilic. This surface property regulates adsorption of proteins over the surface of the biomaterial. Thus, little amounts of proteins will be adsorbed over the bioplastic, when it comes in contact with bodily fluids. Example 5

Quantification of protein adsorption The amount of protein adsorbed on the bioplastic surface vis- a-vis a control surface of tissue culture polystyrene was measured through a method of differences using bovine serum albumin (BSA) as a model protein. Briefly, a two millilitres sample from a stock solution of 50 μg / mL BSA in normal saline was added to each of three 10 cm tissue culture polystyrene petri dishes (control group, triplicate) and similar three 10 cm petri dishes overlaid with a layer of bioplastic (experimental group, triplicate). The solution was swirled in all the six petri dishes to coat the surface, and the petri dishes were kept on a rocker for 20 minutes at 35 °C. The amount of protein in the supernatant solution in both sets of petri dishes was estimated through Bradford assay. Subtracting these values from 100 μg gave the amount of protein adsorbed on the respective surfaces. The amount of protein adsorbed per unit area was calculated by dividing the previous figure by the surface area of the petri dishes. The results are shown in figure 8 which shows the adsorption of bovine serum albumin on agarose based bioplastic of the present invention and tissue-culture polystyrene (TCPS, control) surfaces. The amount of protein adsorbed over the bioplastic was roughly half of that over the tissue-culture polystyrene surface. Since protein adsorption guides host reaction to biomaterial, the bioplastic shall initiate abated host responses when used as a drug delivery vehicle.

Example 6

Direct and indirect contact assays

For direct contact assay, 300 μΐ molten bioplastic was added to cover the bottom surface of six wells of a 24-well tissue-culture polystyrene plate. These wells were then cultured with chicken fibroblasts (DF-la) in serum-fortified DMEM medium at 37 °C for 36 hours in a humidified incubator with 5% carbon-dioxide environment. As a positive control, chicken fibroblasts were cultured under similar conditions in the wells of ultra-low attachment 24- well culture plates. For indirect control assay, 50 μΐ of molten bioplastic was added to the sides of six wells of a 24-well tissue-culture polystyrene plate in such a way that more than half of the bottom surface of these wells remained uncoated. These wells were then cultured with chicken fibroblasts in serum-fortified DMEM medium at 37 °C for 36 hours in a humidified incubator with 5% carbon-dioxide environment. As a positive control, chicken fibroblasts were cultured under similar conditions in other wells of the same plate. These wells did not have any molten bioplastic added to their sides. For negative control of both assays, fibroblast cells were cultured under similar conditions in the wells of a 24-well tissue-culture polystyrene plate, with the exception that 0.1% Triton-X 100, a known non-biocompatible chemical, was added to the wells to induce apoptosis.

At the end of culture period, the cells were observed and imaged in an inverted microscope (Leica DMIL LED) under differential interference contrast settings to investigate their morphology. MTT assay for investigation of metabolic activity of the cells was also performed using Tada's modification of the standard MTT assay. The results are shown in figure 9 which shows direct and indirect contact cell culture assays for assessment of the cytocompatibility of agarose based bioplastic of the present invention. The morphology of cells in indirect contact with the bioplastic (a) closely resembled that of cells cultured directly on adherent surface (b). These cells were well spread over the surface, and revealed the classic morphology of fibroblasts. Similarly, the morphology of cells in direct contact with the bioplastic (c) mirrored that of cells cultured directly on a non-adherent surface (d). These cells were either unattached to the surface or revealed spherical morphology without any spreading. All these were starkly different from the morphology of dead cells (e). These divulged shrunken morphology typical of apoptotic cells. While dead cells did not show any metabolic activity in MTT assay, the metabolic indices of cells cultured in direct and indirect contact with the bioplastic resembled those of their respective positive controls (f). These assays showed that the bioplastic did not produce any morphological or metabolic changes in the cells under our experimental conditions. Example 7

Study of haemolyticbehaviour

The study of haemolytic ability of the agarose based bioplastic of the present invention was investigated using mouse blood. The collection of blood cells was performed as follows. Two millilitres of blood collected from freshly sacrificed healthy female mice was diluted 1:25 with phosphate buffered saline pH 7.4 to alleviate the effect of clotting factors. The blood cells (predominantly erythrocytes) in the suspension were collected by centrifugation at 150 xg for 10 minutes. After removal of the supernatant, the cells were re-suspended in 5 millilitres of phosphate -buffered saline pH 7.4. Haemolysis was performed in 2 mL microcentrifuge tubes. The negative control group had 200 μΐ. PBS, while the positive control group had 200 \xL 2% Triton-X 100 detergent. 200 μΐ. of molten bioplastic solution was added to the experimental group, and allowed to solidify. To all these microcentrifuge tubes, 800 \xL of blood cell suspension was added, and these were then incubated at 37°C for 90 minutes. In the end, these micro-centrifuge tubes were centrifuged at 150 x g for 10 minutes to pellet cells, and the absorbance of the supernatant at 541 nm measured for each in a UV-Vis spectrophotometer (Thermo Scientific Evolution 201). The percentage of haemolysis was calculated as:

. Abssr emse of sa ple

HsemolTsis {_%)— ^; x 100

AbsQT&ance of -positiife controi

The results are shown in figure 10 which shows haemocompatibility assay of agarose based bioplastic sample vis- a-vis phosphate -buffered saline pH 7.4 (negative control) and 2% Triton- X 100 (positive control). The percent haemolysis seen in the bioplastic sample was negligible, similar to that seen in the negative control. In the positive control, large amount of haemolysis occurred. The results indicate that the agarose based bioplastic is highly haemocompatible. Example 8

Study of drug release rate

Films, made of agarose based bioplastic of the present invention, loaded with 100 μg / mL of ampicillin were prepared. From these small samples were cut such that each sample had 60 μg of ampicillin. These samples were immersed in phosphate -buffered saline solution, pH 7.4 for pre-decided periods of time, in different tubes at 37°C. The peak absorbance of ampicillin is known to be at 220 nm. After the designated time had elapsed, the supernatant was removed and its absorbance was recorded at 220 nm using a UV-Vis spectrophotometer (Thermo Scientific NanoDrop ND-1000). Average of three readings was recorded, and the readings converted to ampicillin concentrations by comparing them with absorbance readings of ampicillin standard curves prepared using known concentrations of ampicillin in phosphate-buffered saline, pH 7.4. The results are shown in figure 11 which show representative curve for the rate of release of ampicillin from these films. The linear range of standard curve for ampicillin (a) and glycerol (b) was used to determine the amount of ampicillin released by bioplastic discs impregnated with 60 g ampicillin into phosphate- buffered saline pH 7.4 at 37°C (c). Since maximum possibleglycerol concentration resulting from the films was less than 0.5%, the corresponding absorbance resulting from the contribution of glycerol was insignificant when compared with the absorbance readings resulting from ampicillin. Thus, the absorbance readings from ampicillin-impregnated agarose basedbioplastic films could directly be used for the quantification of ampicillin, without making an explicit quantification of the amount of glycerol released from the films. The curve (c) showed that the release of ampicillin is complete in the first hour.

Example 9

Study of drug potency Circular samples of 10 mm diameter were cut from drug-loaded films of example 8. The concentration of antibiotics in agarose solution was 100 μg / mL, while Dettol® and Savlon® were diluted to 1 in 20 and 1 in 15 concentrations, respectively, as suggested by the respective manufacturers. Samples cut from control films did not contain any drug. 300 million colony forming units of Escherichia coli (strain: DH5a) were spread on LB agar plates. The cut samples of the drug loaded films were placed on top of these plates, and the plates were incubated at 37°C for 12 hours. The maximum diameter of the zone of inhibition was measured for each film, and images were recorded. The results are shown in figure 12 which shows study of potencies of drugs incorporated in the films, as determined through agar diffusion assay. The agar-diffusion assays wereperformed on control films without any antibacterial drugs (a), and on films loaded with ampicillin (b), kanamycin (c), tetracycline (d), dettol (e) and savlon (f). The films loaded with drugs had a distinct zone of inhibition around them where bacterial growth was impeded, whereas the control films did not hamper the growth of bacteria. The sizes of the zones of inhibition for the drugs are also depicted (g).

Example 10

Fabrication of fiber-reinforced bioplastic with bacterial cellulose

The fabrication of agarose based bioplastic is described in Example 1. To cast the films, twenty milliliters volume of the bioplastic mixture was poured into 90 mm petri plates, cooled and dried at 25 °C for 72 hours at 50% relative humidity.

Bacterial cellulose reinforced composites were fabricated by adding different volumes of the slurry of homogenized bacterial cellulose into the agarose based bioplastic to give a final cellulose concentration of 0, 10, 15, 20, 25, 30, 40 and 50% (w/w) with respect to the agarose. Films with stained cellulose fibers were also produced to study the dispersion of fibers in the agarose based bioplastic matrix.For mixtures containing cellulose in excess of 30% (w/w with respect to agarose), the solution became so viscous that it could not be casted. Example 11

(a) Mechanical testing of the composite bioplastics

The thicknesses of the various filmsprepared in Example 10 were measured using Digimatic micrometer (Mitutoyo Corporation, Japan) and were found to lie between 150 and 400 μπι. The thickness of the films increased with higher amount of cellulose. ASTM D 1708-59 T standard was followed to measure the tensile properties of the samples. The samples were cut into dog-bone shaped specimens using EpilogLaser C02 laser engraving and cutting machine. As per standard, they were incubated at 25°C and 50% relative humidity for 72 hours, and then tested on Instron 3345 tensile testing machine with an initial grip separation of 0.9 inches (22.86 mm) and crosshead speed of 1 mm/min as per the above ASTM standard. Multiple specimens were tested for each sample, and the average ultimate tensile strength was recorded as illustrated in Figure 13.

The tensile strength of agarose based bioplastic (25.1 MPa) increased with adding bacterial cellulose to a maximum at 60.2 MPa for 20% cellulose [Figure 13].

(b) Effect of reinforcement on swelling properties

Swelling behaviour of bioplastic films at three temperatures, 4°C, 25°C and 37°C, was studied as follows. Water baths containing deionized water for each of the above temperatures were set. Small strips of bioplastic films (size 10 mm x 30 mm) were cut and weighed. A strip of the bioplastic film was immersed in each water bath. After every 15 minutes, the strips were taken out, wiped with filter paper, weighed and then put back into their respective water baths. The measurements were continued for a little over two hours, by when the successive readings had equalized. The percentage of swelling (Sw), defined by the equation:

Weight after swe ina— initial- weight was plotted as a function of time elapsed.

Agarose based bioplastic absorbs large quantities of water, in excess of 700% of its weight at 37°C. Addition of cellulose to the bioplastic led to a marked reduction in the amount of water absorbed [Figure 14]. For bioplastic films with 20% cellulose (w/w with respect to agarose), the amount of water absorbed at 37°C was around 450% of the weight of the film. Similar trend was also observed at 4°C and 25°C where reinforced bioplastic swelled to a lesser extent than the virgin bioplastic.

Example 13

Preparation of cross linked agarose based bioplastics

Crosslinker (10% citric acid and 5% sodium hypophosphite, w/w with respect to agarose) were added to molten agarose based bioplastic of Example 1. The solution was swirled manually again, before casting into films. The bioplastic films were thoroughly dried in a vacuum desiccator lined with anhydrous silica gel for 5 days. Crosslinking was done in a hot air oven. The films were crosslinked at 110°C for various time, ranging from 0 to 25 minutesbefore being removed and cooled to room temperature. After the reaction was complete, the films were incubated at 25°C and 50% relative humidity for three days, and their strength measured following ASTM D 1708-59 T standard.

Samples of the films were driedfor five days in a vacuum desiccator lined with anhydrous silica gel, before being ground into a powder. FTIR spectra of the powders were taken using Perkin Elmer Spectrum Two spectrometer in the wavenumber range from 400 cm^"1 to 4000 cm^"1 as illustrated in Figure 15.

Example 14

(a) Effect of crosslinking on ultimate tensile strength

The thicknesses of the films were measured using Digimatic micrometer (Mitutoyo Corporation, Japan) and were found to lie between 140 and 256 μπι. The thickness of films varied with changing acid concentration. ASTM D 1708-59 T standard was followed to measure the tensile properties of the samples. Briefly, the samples were cut into dog-bone shaped specimens using EpilogLaser C0₂ laser engraving and cutting machine. They were incubated at 25°C and 50% relative humidity for 72 hours, and then tested on Instron 3345 tensile testing machine with an initial grip separation of 0.9 inches and crosshead speed of 1 mm / min as per the above ASTM standard. Specimen was tested for each sample, and the average ultimate tensile strength was recorded as illustrated in Figure 16.

Their tensile strength of 52.7 MPa was nearly twice the tensile strength of non-crosslinked control films (Strength: 25.1 MPa).

(b) Effect of crosslinking on swelling properties Swelling behaviour of bioplastic films at three temperatures, 4°C, 25°C and 37°C, was studied. Water baths containing deionized water for each of the above temperatures were set. Small strips of bioplastic films (size 1 cm x 3 cm) were cut and weighed. A strip of the bioplastic film was immersed in each water bath. After every 15 minutes, the strips were taken out, wiped with filter paper, weighed and then put back into their respective water baths. The measurements were continued for a little over two hours, by when the successive readings had equalized. The percentage of swelling (Sw), defined by the equation:

Weight after swelling— Initial weight;

= : — tOO

Init weig t was plotted as a function of time elapsed.

Crosslinking resulted in a drastic reduction in the amount of water absorbed in swelling experiments; for instance, the swelling percentage reduced from 700% to 80% of the weight of the film at 37°C as illustrated in figure 17.

(c) Effect of crosslinking on degradation behaviour

Degradation in formic acid was done at 30°C. 200 mg samples from crosslinked and control films were put in 20 g formic acid in polypropylene tubes for 30 minutes and 5 hours. After the specific times, the films were removed from formic acid, rinsed in deionized water, dried in a hot air oven set at 60°C for four days, and weighed. The difference between the initial weight (200 mg) and the final weight gave the amount degraded, which was converted to percentage. The average of three readings was recorded as illustrated in Figure 18. (d) Thermogravimetric analysis of crosslinkedbioplastic

Thermogravimetric analysis (TGA) was performed on SDT Q600 equipment from TA instruments. The films were dried in a vacuum desiccator lined with anhydrous silica gel for three days. 10 mg samples of the films were cut, and the TGA performed in an inert atmosphere of nitrogen gas with a purge rate of 100 mL per minute, with temperatures rising from ambient conditions to 700°C at the rate of 10°C / minute. In between, the samples were held at 80°C for 10 minutes to remove any absorbed moisture.

While the films were completely degradable in formic acid, the rate of degradation was slower for crosslinked films. Thermogravimetric analysis revealed that the rate of thermal degradation of crosslinked films is also lesser than that of control films as illustrated in Figure 19.

Example 15

Incorporation of silver in agarose based bioplastics

These agarose based bioplastic films were loaded with silver at 1 mM concentration, utilizing their swelling ability. In brief, the bioplastic films were dried followed by soaking in AgN0₃ solution and then dried again. The drying steps were done in a vacuum desiccator lined with anhydrous silica gel for three days. Silver nitrate in the films was reduced by a freshly prepared NaBH₄ solution. The soaked films were removed and dried at 25 °C for 72 hours at 50% relative humidity.

Example 16 Scanning electron microscopy and energy-dispersive X-ray spectroscopy Bioplastic film samples were dried by placing them with anhydrous silica gel in a vacuum desiccator for three days. ZEISS EVO50 Scanning electron microscope with Oxford Instruments EDS attachment (Model no. SIXMX1118) was used for characterization of film samples. The films were coated with a layer of gold through sputtering before characterization. The EDS was done at an accelerating voltage of 20 kV as illustrated in Figure 20.

Example 17

Anti-bacterial properties of silver nanoparticle incorporated bioplastic films

The anti-bacterial property of the bioplastic films was tested through a variation of the standard agar diffusion assay. Briefly, overnight cultures of DH5aE. coli bacteria were spread on LB-agar plates. Dilution counts showed that each plate received 270 million colony-forming units of bacteria. Square samples of size 1 cm x 1 cm were cut from the silver nanoparticle loaded and control bioplastic films, and were laid on top of the bacterial plates. These were then incubated at 37°C for 8 hours. The plates were then taken out and imaged to study the zones of inhibition, if any, in the plates as illustrated in Figure 21.

Claims

1. A process for preparation of a bioplastic material, said process comprising steps of a) mixing and heating of agarose with v/ater and a plasticizer to boiling temperature of 90°C to 100°C; b) cooling of solution from step (a) to a temperature of about 60°C; and c) castingsolution from step (b) into sheets wherein the amount of the plasticizer ranges from 1 to 50% w/w;

2. The process as claimed in claim 1 , wherein said agarose is a purified agar processed to have only straight linear chains.

3. The process as claimed in claim 1, wherein said plasticizer is glycerol.

4. The process as claimed in claim 1 , wherein said plasticizer is present in an amount of 20%w/w.

5. A bioplastic material prepared by the process of claim 1.

6. A bioplastic materialas claimed in claim 5, wherein said bioplastic possesses tensile strength ranging from 66MPa to 20 MPa.

7. The bioplastic material as claimed in claim 5, wherein water makes an average contact angle of about 11.4°on said bioplastic.

8. The process for preparation of bioplastic material as claimed in claim 1, wherein said process comprises a further step of adding bacterial cellulose in a concentration ranging from 10 to 30% w/w of agarose.

9. The process for preparation of bioplastic material as claimed in claim 1, wherein said process comprises a further step of crosslinking with citric acid in presence of sodium hypophosphite at 110°C for 15 minutes.

10. The process as claimed in claim 11, wherein the concentration of citric acid is about 10% w/w of agarose.

11. The process as claimed in claim 11 , wherein the concentration of sodium hypophosphite is about 50% w/w of citric acid.

12. The process of preparation of bioplastic as claimed in claim 1, wherein said process comprises a further step of imbibing silver nitrate and sodium borohydride at a temperature of about 25°C and for 72 hours.

13. An article including the bioplastic material as prepared by the process claimed in the preceding claims.

14. An article as claimed in claim 13, wherein said article is a drug delivery vehicle.