In Vitro Cytotoxicity of Amylose-Based Bioplastic for Packaging Applications
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Abstract
Amylose containing polysaccharides are one of the most abundant and inexpensive naturally occurring biopolymers. Therefore, they are one of the most promising candidates to produce substitute plastics, especially in packaging applications. To determine the suitability for packaging applications, cytotoxicity of a modified amylose based bioplastic was investigated using NIH 3T3 Fibroblast cells from observation of cell morphology and MTS assay. Chemical durability of the amylose based bioplastic film was also studied by ion release and pH measurement after immersing the film into water. In vitro cytotoxicity (Cell morphology study and MTS assay) showed that the amylose based bioplastic film has in vitro biocompatibility and can be used for packaging applications. The ion release and pH measurement also supported the results.
Keywords
Amylose Biopolymer Bioplastics Biodegradable Biocompatibility Cytotoxicity PackagingIntroduction
More restrictive regulations, increasing waste disposal costs and threats of uncertain petroleum supply have resulted into renewed interest for alternative solutions. Biodegradable plastics made from renewable resources provide a sustainable alternative to the conventional petroleum-based plastics. The use of plastics derived from bioresources would reduce dependence on imported oil and alleviate the problems associated with handling of environmental waste, particularly from discarded petroleum based plastics. There is therefore, a huge demand to make packaging materials from biodegradable plastics. Materials derived from agriculture especially those containing polysaccharides like corn, peas, potato, barley etc are found in abundance and at low cost. It is well known that one of the important fractions in these products is amylose. Therefore, amylose-based polysaccharides have become one of the most promising candidates as alternative materials to replace traditional plastics in certain market segments, such as the food packaging industry. A number of studies have been conducted to optimize the performance of the amylose-based plastics [1, 2, 3, 4]. However, the design and engineering of an amylose-based packaging product is a significant challenge. Numerous studies have aimed to modify the functional properties of the amylose to enhance the inherent bonding strength. Currently, most of the studies are focused on incorporating additives, such as plasticizers, or chemical modification to improve the performance of the material [5, 6].
However, it is important that biocompatibility of modified amylose-based bioplastic be measured before the material is used for food and packaging applications. Cytotoxicity test is the most commonly used form of screening for determining biocompatibility. The measurement of cell proliferation and cell viability has become a key technology in the biocompatibility test. Proliferation assays have become available for analyzing the number of viable cells by the cleavage of colourless or lightly coloured substance added to the culture medium. Tetrazolium salts are attractive candidates for this purpose since the tetrazolium ring is cleaved in active mitochondria, and therefore the reaction occurs only in living cells. The MTS cell proliferation assay is one such colorimetric assay which is designed to be used for non-radioactive, spectrophotometric quantification of cell growth and viability in proliferation and chemosensitivity. The test utilises yellow tetrazolium compound (3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]) which is metabolised by mitochondrial succinic dehydrogenase activity of proliferating cells to yield a purple formazan reaction product that is soluble in tissue culture medium. The assay is therefore, a quantitative measure of mitochondrial activity and thus in turn cell viability or cytotoxicity. Cell viability is also influenced by presence of ions and pH and is therefore required to be investigated.
In this article, the in vitro cytotoxicity of a modified amylose bioplastic film was evaluated by observation of cell morphology under scanning electron microscope and MTS assay measurement. Additionally, ion release and pH studies were conducted to determine the chemical durability of the bioplastic film.
Materials and Methods
Materials
A pre-commercial scale thermoplastic starch film containing modified amylose has been used for the cytotoxicity tests. The material was obtained from the Centre for Biocomposites and Biomaterials Processing (CBBP), University of Toronto. The details of the material properties have been described elsewhere [7]. NIH 3T3 Fibroblast cells were used to determine the cytotoxicity of the bioplastic film. The ‘CellTiter 96® AQueous One solution’ reagent was used for the measurement of MTS assay.
Ion Release and pH
The bioplastic film (2.5 cm × 2 cm) was cut and placed in separate plastic bottles with 30 mL of distilled water. After 10 and 15 days, the bottles were well shaken and amount of ions like Ca, K, Mg, Si and Na were measured using inductively couple plasma-mass spectrometry (ICP-MS). The ions from distilled water were also measured as a control. The amylose film (2.5 cm × 2 cm) was also placed in distilled water for pH measurement. The change of pH after 1 h to 7 days was measured using a calibrated pH meter. The pH of distilled water was measured as a control.
Cell Culture
Film samples (2 mm × 3 mm) were first sterilised by autoclaving (15 min at 121 °C/15 psi). Cytotoxicity was investigated using NIH 3T3 Fibroblast cells. Because, cryopreserved cells are fragile and require gentle handling, fibroblast cells were removed from liquid nitrogen and placed in warm water (37 °C) quickly for thawing. The freezing medium was removed and the cells were washed using sterile PBS (Phosphate Buffered Saline). The cells were then cultured using a supplemented medium (DMEM) with 10% FCS (Foetal Calf Serum) and 1% Penicillin/Streptomycin/Glutamine. Cells were kept inside an incubator at 37 °C in a 5% CO2 atmosphere to populate the surface of culture plates. Cell splitting was carried out using Trypsin-EDTA. Split interval and number of passages were decided based on a number of factors such as cell proliferation and differentiation rate, cell confluency, health and morphology. Based on these variants, the splitting process was carried out after every 48–72 h until enough seed density was achieved for further culture.
For scanning electron microscopy (SEM), the cells were seeded into wells of a 24 well plate containing test samples (12 mm diameter film) with a total volume of media of 500 μL. The materials and cells were incubated at 37 °C in a 5% CO2 atmosphere for 24 h. NIH 3T3 Fibroblast cells grown on glass cover slip were considered as control. After culturing of cells the samples and cover slip were taken out and fixed, dehydrated, sputter-coated with gold and observed using scanning electron microscopy.
For MTS assay, the cells were seeded into wells of a 96 well plate containing test samples (seeding density of 3 × 104 cells/mL) with a total volume of media of 500 μL. The materials and cells were incubated at 37 °C in a 5% CO2 atmosphere for 72 h. NIH 3T3 Fibroblast cells grown on tissue culture plastics were considered as control. Tissue culture plastics had been treated chemically to promote cell adhesion. Assays were performed by adding a small amount (20 μL) of the ‘CellTiter 96® AQueous One solution’ reagent directly to culture wells containing 100 μL of culture media and the plate was left to incubate at 37 °C for 3 h. The intensity of the coloured solution was measured using a spectrophotometer at a wavelength of 490 nm.
Results and Discussion
Ion Release and pH
Amount of ions released from amylose film after immersion in water
Ions | 10 Days mean (mg/L) | 15 Days mean (mg/L) |
---|---|---|
Ca | 0.699 ± 0.0029 | 0.279 ± 0.0014 |
K | 1.23 ± 0.004 | 5.45 ± 0.024 |
Mg | 0.191 ± 0.0007 | 0.052 ± 0.0006 |
Na | 0.531 ± 0.0012 | 1.08 ± 0.003 |
Si | 0.016 ± 0.0010 | 0.004 ± 0.0001 |
As | 0.060 ± 0.0114 | 0.017 ± 0.0019 |
Zn | 0.011 ± 0.0007 | 0.064 ± 0.0016 |
Change of pH with time of amylose bioplastic film immersed into distilled water
Cell Morphology
a Scanning electron photomicrograph showing NIH 3T3 Fibroblast cells after 24 h culture on the surface of bioplastic film. b Scanning electron photomicrograph at higher magnification showing NIH 3T3 Fibroblast cells after 24 h culture on the surface of bioplastic film. c Scanning electron photomicrograph showing NIH 3T3 Fibroblast cells after 24 h culture on glass cover slip as a control. d Scanning electron photomicrograph at higher magnification showing NIH 3T3 Fibroblast cells after 24 h culture on glass cover slip as a control
MTS Assay
Bar chart showing MTS assay results
Scoring system for estimating cytotoxicity in MTS assay
Cytotoxicity index | 0 | 1 | 2 | 3 | 4 | 5 |
---|---|---|---|---|---|---|
RGR (%) | >100 | 75–99 | 50–74 | 25–49 | 1–24 | 0 |
For cytotoxicity index ranging from 0 to 1, the materials are considered to pass the cytotoxicity test. For index 2, it is advised to repeat the test, or evaluate in combination with the cell morphology observation. For index reading between 3 and 5, the material is considered to fail the test. Relative growth ratio (RGR) of cells for amylose bioplastic was calculated as 75.8. Based on that, the cytotoxicity index of the bioplastic is 1 and the material is considered to be non-cytotoxic. Quantitative MTS assay result from modified amylose-based bioplastic therefore, certainly strengthens the qualitative SEM observation and indicates no or minimal cytotoxicity of this material.
Conclusions
This study clearly demonstrates that modified amylose-based bioplastic film is non-cytotoxic. The bioplastic film did not release any significant amount of ions after immersion in distilled water and there was no considerable change in pH values with time. From the cell morphology study and the MTS assay, it is clear that this modified amylose based bioplastic film shows in vitro biocompatibility with fibroblast cells and they had no or minimal cytotoxicity. While further studies are required to get a clear idea about the structure property relationship, this study clearly suggests that this modified amylose-based bioplastic film have significant potential for packaging applications.
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