In vitro biostability and biocompatibility of ethyl vinyl acetate (EVA) nanocomposites for biomedical applications

Abstract

The in vitro biostability and biocompatibility of ethyl vinyl acetate (EVA) nanocomposites incorporating organically modified montmorillonite (organo-MMT) were investigated as new candidate material for biomedical applications. The in vitro treatment of neat EVA and EVA nanocomposites was performed by immersing the materials in oxidizing and hydrolytic agents, at a temperature of 37 °C, for 4 weeks. The in vitro mechanical properties of the materials under these environmentally challenging conditions were assessed. Based on morphology studies, the degree of MMT dispersion and exfoliation decreased as the nanofiller loading increased. The EVA containing 1 wt% organo-MMT exhibited the best nanofiller dispersion and exfoliation characteristics. The surface degradation features of this nanocomposite were seen to be smoother than those of neat EVA and other EVA nanocomposites. Furthermore, the EVA nanocomposites have improved mechanical properties in comparison with the neat EVA, and these properties were less affected by the in vitro conditions. The best in vitro mechanical properties were achieved when 1 wt% of organo-MMT was added into the EVA. It was postulated that the presence of a better dispersed and exfoliated organo-MMT layered structure introduced a more tortuous path for the diffusion of oxidants and water molecules, thereby decreasing their permeation towards the EVA molecular chains. Therefore, the degradation kinetics within the EVA molecular chains were at a lower rate, which resulted in enhanced biostability. Furthermore, the toughness of the hydrated EVA (exposed to PBS at 37 °C) was greatly enhanced with the addition of the 1 wt% organo-MMT. The biocompatibility assessment suggests that the EVA nanocomposites are not cytotoxic, and thus have fulfilled the prerequisite to be further developed as a biomedical material.

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1. Introduction

In this new era, in which disease and health problems are the main issue in society, there is a constant demand for cost-effective and innovative biomedical materials for both medical devices and packaging purposes. However, a problem in the medical field is the limited number of existing biocompatible, biostable and tough materials that offer versatility and exceptional performance, and that can feasibly be produced with current industrial methods. This is likely to be due to the stringent requirements in place, regarding the materials’ properties, design and processing, as well as the constraints on their availability.^1,2 The increasing research and development of quality plastic materials is therefore vital in order to fulfil the requirements of this diverse medical industry. This is believed to be an area where innovation in new medical nanocomposite materials is needed. Biocompability is a prerequisite for any materials intended for use in biomedical device applications.^1,3 The application of polymer–clay based nanocomposites as biomedical materials is hampered, in particular, due to their unknown biosafety. The organic surfactants used as surface modifiers could compromise the biocompatibility of these materials.^4–6 For long term safety, the materials should possess biostability, to withstand repeated sterilization processes that may involve gamma irradiation, high temperatures, electron beams, and oxidative and hydrolytic treatments.³ In addition, a material must show excellent chemical resistance, toughness, clarity, and color stability in order to be effectively applied in biomedical applications.^1–3 Meanwhile, for the medical packaging purpose, the material should withstand prolonged mechanical stress and impact forces during transportation and storage. Therefore, the recommended properties are strength, toughness, durability, tear resistance and the ability to protect the packaged contents from physical damage.^7,8 The absence of these properties may result in the occurrence of pinholes in the structure of the packaging materials, especially during transportation and long storage. In the worst case scenario, this pinhole defect can allow the entrance and accumulation of bacteria on the medical device and bring a health risk to patients. Achieving an effective biomedical nanocomposite material that meets these stringent criteria may be accomplished by carefully examining the structure, morphology and mechanical performance of this material under ambient and environmentally challenging conditions.

Ethylene vinyl acetate (EVA) is the copolymer of ethylene and vinyl acetate. EVA combines the chemical and material properties of a chemically cross-linked elastomer with those of engineered plastics, which are often much easier and affordable to manufacture. It is an extremely elastic material that can be sintered to form a porous material similar to rubber, yet with excellent toughness.⁹ EVA has a long and successful history as an innovative material in medical packaging, medical devices, and pharmaceutical applications. In fact, EVA has been an innovative material in those applications for over 35 years and its wide range of properties can be tailored and manipulated to develop novel materials with numerous potential applications.^9–11 For biomedical applications, the continued research and innovation based on this material are required for the continual improvement of patient healthcare devices and medical support.

EVA nanocomposites are a relatively new class of materials that leverage the benefits of engineered plastics, the nanofiller and elastomeric properties. The incorporation of a small amount of nanometer sized particles can significantly affect the final properties of the EVA and offer a tremendous improvement in its physical and mechanical performance.^12,13 A review of the current literature suggests that there is only limited research on the development of EVA–nanoclay based nanocomposites for biomedical applications. Most of the work focuses on the investigation of the EVA–nanoclay nanocomposites in general film packaging and flame retardant applications.^12–17 The next generations of medical devices and medical packaging require materials that are very biostable, easily processed and have substantially improved mechanical performance.^1,3 The well-formulated EVA–MMT nanocomposites could be potential candidates for several medical/pharmaceutical devices and packaging applications, including thermoformed trays, containers, boxes, tubes, needle covers, bottles, blister packs, clamshells and bottle caps. The exploration and thorough study of EVA nanocomposite systems is therefore needed to develop this versatile material into innovative products. The targeted properties can be achieved through careful formulation of the EVA nanocomposite, and optimized EVA–nanofiller interactions. In an effort to achieve this goal, we have performed investigations on the structure and properties of the EVA/MMT nanocomposites under ambient and in vitro conditions. Furthermore, a preliminary biocompatibility evaluation was also done as part of assessments to determine their suitability for use in a broad range of biomedical applications. Some morphology and preliminary in vitro biostability and biocompatibility studies are reported and discussed herein.

2. Experimental section

2.1. Materials

The matrix material used was ethyl vinyl acetate (EVA), which is a thermoplastic copolymer and solid at room temperature. It is manufactured by UBE-Maruzen Polyethylene Co. Ltd., Tokyo, Japan and commercially known as UBE EVA V215. The weight percentage of vinyl acetate is 15%, with the remainder being ethylene. Organically modified montmorillonite (organo-MMT), which contains 35–45 wt% dimethyl dialkyl (C14–C18) amine as an organic surfactant, was used as the nanofiller. It was manufactured by Sigma-Aldrich (USA) and supplied by Zarm Scientific and Supplies Sdn. Bhd. It is a beige coloured powder with the chemical formula (Na,Ca)_0.33(Al,Mg)₂(Si₄O₁₀)(OH)₂·nH₂O. The average particle size of this organoclay is ≤20 microns, while the bulk density ranges from 200 to 500 kg m⁻³. The oxidizing agent used in this research was hydrogen peroxide (H₂O₂). H₂O₂, 30–32% solution (Qrec®), was supplied by Qrec (Asia) Sdn. Bhd. Phosphate-buffered saline (PBS) tablets were manufactured by Sigma-Aldrich and utilized as the hydrolytic agent after being dissolved in distilled water.

2.2. Preparation of the samples

The samples were prepared by melt compounding the EVA copolymer with different ratios of organo-MMT nanofiller (0, 1, 3, and 5 wt%) using a Brabender Plasticorder machine (manufactured by Lab Tech Co. (LZ80)). Firstly, the materials (EVA and organo-MMT) were dried in the oven at a temperature of 50 °C for 24 hours. After the temperature in the Brabender Plasticorder machine reached 160 °C, the EVA pellets were fed into the feeder and allowed to melt for 4 minutes. Then the organo-MMT was added and compounded with the EVA melts for a further 6 minutes. The resulting nanocomposite samples were cut and weighed to about 20 g and then compressed into 1 mm thick sheets using the compression moulding machine model GT-7014-H30C manufactured by GOTECH Co. The temperature of this process was set at 160 °C. The samples were compressed at 10 kPa for 3 minutes, and then cooled under pressure for another 3 minutes. The samples were then cut accordingly for testing and analysis.

2.3. Characterization and mechanical testing

2.3.1. X-ray diffraction (XRD) analysis. XRD analysis of the organo-MMT nanofiller and nanocomposites was done using the XRD device model Phaser-D2 manufactured by Bruker, to characterize basal plane spacing changes in the organo-MMT, due to the insertion of EVA molecular chains within the layered structure.

2.3.2. Transmission electron microscopy (TEM). The dispersion of the organo-MMT inside the EVA matrix was assessed using TEM. Thin sections approximately 30–50 nm in thickness were cut using a DiATOME diamond knife on a Leica Ultracut microtome (Leica Biosystems GmbH) and placed on carbon copper grids. Samples were examined at low magnification (12 000×) and high magnification (93 000×) on a LEO 922 A EFTEM (Carl Zeiss AG) operating at a 200 kV acceleration voltage.

2.3.3. Scanning electron microscopy (SEM). The surface degradation of the neat EVA and EVA nanocomposites was investigated using SEM (Leo 1530 FESEM Zeiss), before and after 4 weeks of immersion in H₂O₂ solution at 37 °C.

2.3.4. Water permeability test. A water permeability test using PBS solution was done to study the hydration characteristics of the studied materials and relate it to their in vitro biostability. Rectangular test samples with dimensions of L = W = 3 cm were cut, oven dried for 1 hour to remove the moisture and immediately weighed to determine the initial mass. Next, the samples were immersed in a beaker containing phosphate-buffered saline (PBS) solution and kept at 37 °C, which is approximately human body temperature. The sample mass was recorded every 7 days after immersion. The hydration characteristics of each material were determined from the percentage increase in mass after each week of immersion, using the following calculation:

Increase in mass (%) = (Hydrated mass − Initial mass) × 100/Initial mass (1)

2.3.5. Ambient and in vitro tensile tests. The preliminary studies on the in vitro mechanical properties were carried out as a first step towards predicting the potential improvements in the long-term in vivo performance of the EVA nanocomposites for long-term medical device applications. The tensile tests on the neat EVA and EVA nanocomposites were performed using an Instron machine model-5582, before and after being immersed in PBS (hydrolytic agent) and H₂O₂ (oxidative agent) solutions, to study the effect of exposure to hydrolytic and oxidative agents on the tensile properties of these materials. The ASTM D638 method was used, and ten replicates of dumbbell samples from each material were tested. Mean values for tensile strength, elongation at break and toughness were taken for the comparison of the materials.

2.4. Biocompatibility test

The Chang liver cell line was used to assess the cytotoxicity of the neat EVA and EVA nanocomposites, due to its ability to indicate the hepatotoxicity of the polymeric materials when they are incubated together. Since the liver is an important target of toxic leachable substances and xenobiotics, and site of oxidative stress, this hepatotoxicity assessment is considered a very sensitive means of testing biocompatibility and biosafety.

Furthermore, this method was chosen due to its lower cost, relatively well-controlled variables and as it yields quantitative results in short time periods.¹⁸

2.4.1. Cell culturing. Chang liver cells were first cultured in a culture medium containing the minimum essential medium (Gibco) in Earle’s BSS with non-essential amino acids and 1 mM sodium pyruvate with 10% fetal bovine serum. Trypsinization and subcultivation of the cells were done by following the standard procedures.
2.4.1.1. Co-cultivation of Chang liver cells on the surface of EVA and EVA nanocomposites: direct contact toxicity assay. The cytotoxicity assessment was performed by using a direct contact toxicity assay. Chang liver cells (2 × 10⁵) were grown on the surface of neat EVA and EVA nanocomposites (incorporating 1%, 3% and 5% organo-MMT). As for the negative control, Chang liver cells were grown on glass cover slips. All treatments were conducted in a 6-well plate (SPL, Korea) for 96 hours and the cells were incubated at 37 °C in a 5% CO₂ humidified incubator.

2.4.2. Scanning electron microscopy (SEM) analysis for Chang liver cells observation. After 96 hours of incubation time, the culture medium was replaced with warm 4% glutaraldehyde in phosphate-buffered saline (PBS) and fixed for 30 minutes. The Chang liver cells attached on the surface of neat EVA, EVA nanocomposites and cover slips were then post-fixed in 1% osmium tetraoxide at room temperature for 90 minutes. After post-fixation, these materials and cover slips with the attached liver cells were rinsed with PBS solution twice and then the dehydration process was continued using a graded series of ethanol from 10% to 100%. The specimens were rapidly transferred to a critical point drying device (BAL-TEC 030) using liquid carbon dioxide. After the critical point drying process, all of the specimens were attached to 13 mm aluminium stub ducting paint and coated with gold using the JEOL JFC-1600 Auto Fine Coater ion-sputtering device prior to SEM analysis (JEOL JSM-6360LA, Analytical SEM).

3. Results and discussion

3.1. XRD analysis of the organo-MMT and EVA nanocomposites

According to Tettenhost and Roberson,¹⁹ MMT has large repeating inter-gallery distances, especially when complexed with organic compounds and, therefore, basal spacings are evident at small diffraction angles. Basal spacing shifts are more pronounced at small diffraction angles.¹⁹ Therefore, in this study, we focus on the XRD analysis between the 2θ angles of 1.5° and 12°. Fig. 1 shows the XRD patterns of the organo-MMT nanofiller, neat EVA and the EVA nanocomposites containing 1, 3 and 5 wt% MMT. Basal spacings were determined using Bragg’s law (nλ = 2d sin θ).

Fig. 1 XRD patterns of the organo-MMT and EVA nanocomposites containing 1, 3 and 5 wt% organo-MMT.

The XRD profile of the neat EVA shows an amorphous halo. This is expected, as this copolymer does not show any diffraction peaks between 2θ = 0.5° to 10°.²⁰ Thus, the diffraction peaks revealed by the EVA nanocomposites in this 2θ range can be associated with the nanofiller basal spacings. The organo-MMT nanofiller exhibits a (d₀₀₁) basal spacing of 2.7 nm. However, there were changes in the basal spacing and intensity of the XRD patterns after the incorporation of the organo-MMT into the EVA matrix. These could be associated with the nanofiller loading, average platelet size, orientation, degree of inter-platelet registration, platelet/tactoid alignment, and intercalation by the host polymer.^21–26

It is observed that when the 1, 3, 5 wt% organo-MMT was dispersed in the EVA matrix, the XRD peaks were shifted to lower 2θ angles, which is an indication of the increasing inter-gallery spacing between the clay layers, most probably due to EVA intercalation. All of the nanocomposites (with 1, 3 and 5 wt% MMT) exhibit three well-defined diffraction peaks centered at 2θ = 2.4°, 2θ = 4.7° and 2θ = 6.8°, corresponding to basal spacings of approximately 3.8 nm, 1.9 nm and 1.3 nm. However, a decrease in the filler loading (from 5 to 1 wt%) resulted in the lowering of the XRD peaks’ intensities, probably due to better EVA intercalation between the platelets, which produced smaller tactoids. For instance, the nanocomposite with 1 wt% MMT produced the weakest XRD signals, which can be associated with the better quality of the MMT dispersion and intercalation in the EVA matrix in comparison with the other systems. The increase in filler loading resulted in more intense diffraction peaks, which can be associated with the presence of larger tactoids. Bear in mind that in some cases the XRD signature does not differentiate between increased spacing, very small platelets and platelet misalignment, thus misleading results sometimes cannot be avoided. Therefore, the XRD data need to be coupled with the TEM analysis for a more complete assessment of the nanofiller dispersion in the polymer matrix.^21,25,26 In this study, the TEM analysis was used to support the XRD analysis and will be discussed in the next section.

3.2. TEM analysis of the EVA nanocomposites (dispersity analysis)

TEM images of the EVA nanocomposites containing 1, 3 and 5 wt% organo-MMT are displayed in Fig. 2. In general, well dispersed organo-MMT was seen to be distributed throughout the EVA matrix. The EVA nanocomposites incorporating 1 and 3 wt% MMT exhibited a mixed morphology of exfoliated/intercalated clay layers, whereas the EVA nanocomposite containing 5 wt% MMT exhibited mainly an intercalated morphology. All of the nanocomposites contained some large silicate tactoids (up to hundreds of nm in size) dispersed inside the EVA matrix. This shows that the shear energy obtained from the Brabender mixer was not sufficient to peel some of the platelets from the well-intercalated tactoids. The large organo-MMT stacking platelets (tactoids) with limited mobility and a high degree of spatial restriction were also believed to experience inadequate orientational freedom in the matrix, thus making it more difficult for the intercalated polymer to delaminate them into single layers.^25–27

Fig. 2 TEM micrographs of EVA nanocomposites containing 1, 3 and 5 wt% organo-MMT.

It is noticeable that the extent of exfoliation/intercalation decreases as the organoclay concentration increases, which is in good agreement with the XRD results. Larger tactoids were observable in the EVA containing a higher organo-MMT loading. This might be due to the collision between the nanoclay platelets that increases with the concentration, thus leading to platelet agglomeration.^23,28 This suggests that, if the nanoclay loading were to be increased to a higher amount, a greater degree of agglomeration would occur. As a consequence, the exfoliated structure would not be achieved, resulting in the possible worsening of the mechanical performance of the nanocomposites. This will be further discussed in the following section. It is also interesting to observe the variations in the shape, size and geometry of the nanoclay platelets that were distributed throughout the EVA matrix. These variations are likely to be due to the processes of chemical treatment of the surface, melt compounding and compression moulding that can extend the range of shapes and sizes of the organo-MMT.

3.3. SEM analysis of the EVA and EVA nanocomposites (surface degradation upon exposure to an oxidative agent)

SEM characterization was performed for all samples before and after four weeks of exposure to an oxidative agent. The SEM images obtained and displayed in Fig. 3 show signs of surface degradation in the neat EVA and EVA nanocomposites, as a result of exposure to the oxidative agent. The surface of the neat EVA sample was smooth and featureless initially, but after being exposed to H₂O₂ a rough surface with pits and cracks was obtained. On the other hand, the surface degradation features for the EVA nanocomposites containing 1 and 3 wt% MMT were seen to be smoother than those of neat EVA after four weeks of oxidative treatment. Amongst all of the samples, the EVA nanocomposite with 1 wt% MMT displays the smoothest surface. Based on the XRD and TEM results, this material also exhibits the best nanofiller dispersion and exfoliation characteristics. Thus, it can be said that the lesser degree of degradation that took place in this particular nanocomposite was the result of the well dispersed and exfoliated organo-MMT. It was mentioned in previous research that exfoliated and well dispersed organoclay can create a more tortuous path for the entrance of oxidants into the polymer chain, thereby slowing down the degradation kinetics.^29,30 However, when the nanofiller concentration increased, the degradation kinetics also increased due to the poorer and poorer nanofiller dispersion and exfoliation characteristics (see Fig. 2). As expected, the EVA nanocomposite with 5 wt% organo-MMT shows the roughest surface morphology, with larger pits and cracks, due to its more severe degradation (Table 1).

Fig. 3 SEM images of neat EVA and EVA nanocomposites, before and after being exposed to H₂O₂ solution at 37 °C for 4 weeks. The magnification is 5000× (scale bar = 5 μm).

Table 1 Tensile properties of neat EVA and EVA nanocomposites, before and after 4 weeks of in vitro treatment (exposure to H₂O₂ at 37 °C)

Mechanical properties	H2O2 exposure	EVA	EVA + 1 wt% MMT	EVA + 3 wt% MMT	EVA + 5 wt% MMT
Tensile strength (MPa)	Before	12.3 ± 0.7	14.3 ± 0.6	13.0 ± 0.4	12.6 ± 0.1
	After	11.6 ± 0.7	13.7 ± 0.3	12.4 ± 0.2	11.5 ± 0.5
Elongation at break (%)	Before	625 ± 27	687 ± 25	647 ± 16	665 ± 32
	After	613 ± 11	687 ± 31	646 ± 19	607 ± 13
Toughness (MPa)	Before	44.0 ± 3.3	55.7 ± 3.0	49.7 ± 1.0	50.0 ± 1.8
	After	40.8 ± 3.1	53.0 ± 2.9	47.2 ± 0.9	42.6 ± 1.9

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3.4. Ambient and in vitro mechanical properties (exposure to H₂O₂ at 37 °C)

The tensile properties of the neat EVA and EVA nanocomposites (before and after oxidative agent (H₂O₂) exposure) are summarized in Table 2, while their representative stress–strain curves are displayed in Fig. 4. Under ambient conditions, the incorporation of 1, 3 and 5 wt% organo-MMT into the EVA resulted in an increase in tensile strength, elongation at break and toughness. Overall, the highest tensile strength, elongation at break and toughness were achieved when 1 wt% organo-MMT was added, with increases of ∼16.3%, ∼9.9% and ∼26.6%, respectively. However, increasing the nanofiller loading from 1 wt% to 5 wt% resulted in the worsening of these tensile properties, which is most likely due to the poorer organo-MMT dispersion and delamination characteristics, as observed using TEM (Fig. 2) and XRD analysis. It is worth mentioning that the levels of nanofiller dispersion, nanofiller–polymer interaction, nanofiller orientation and nanofiller content are among the factors that determine the tensile properties of the nanocomposites produced.^21–27

Table 2 Reduction in the tensile strength and toughness of the neat EVA and EVA nanocomposites after 4 weeks of in vitro exposure to H₂O₂ at 37 °C

Materials	% reduction in tensile strength after H2O2 exposure	% reduction in toughness after H2O2 exposure
0%	5.7	7.3
1%	4.2	4.8
3%	4.6	5.0
5%	8.7	14.8

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Fig. 4 Representative stress–strain curves for neat EVA and EVA nanocomposites before and after 4 weeks of in vitro exposure to H₂O₂ at 37 °C.

The enhancement in the tensile strength of the polymer nanocomposite is attributed to the reinforcement provided by the dispersed nanoclay platelets. Nanoclay that is better dispersed may promote higher tensile strength due to an increase in the contact surface area and interactions between the platelets and the polymer.^25,31 The well-bonded interface may enable the load transfer and energy dissipation in an area of high stress. This is proved by our results, which show that the best improvement in the tensile strength of EVA was achieved with the addition of 1 wt% organo-MMT, which was also the loading that resulted in the best nanofiller dispersion. The improvement in the toughness of the EVA upon the incorporation of the organo-MMT might be attributed to the plasticizing effect of organic onium ions from the organo-MMT, which affects the conformation of the EVA copolymer chains at the layered platelets–matrix interface. These ions would promote relaxation at local stress regions, allowing the material to achieve a higher elongation at break.³²

Our results show that the incorporation of nanofillers have a greater influence on the stress–strain behaviour of the EVA when exposed to the oxidizing agent (H₂O₂) at 37 °C. The nanocomposites display improved tensile properties when compared with the neat EVA, showing gains in tensile strength, elongation at break and toughness. Interestingly, the nanocomposite incorporating 1 wt% organo-MMT not only displays the best tensile properties, but also retains the most tensile toughness after exposure to the oxidative agent out of all of the materials. As displayed in Table 3, the toughness of the EVA nanocomposite containing 1 wt% MMT was only reduced by 4.8% after 4 weeks of H₂O₂ exposure, while, for the neat EVA, the toughness was reduced more significantly (7.3%). This suggests that the presence of the organo-MMT layered structure may introduce a more tortuous path for the diffusion of oxidant molecules, thereby decreasing their permeation into the EVA molecular chains. The lesser amount of oxidant entering the polymer molecular chains resulted in the greater retention of the nanocomposite’s mechanical properties.^29,30 Toughness is very important for materials intended for biomedical devices, as it will delay crack propagation and lengthen the service of the material. Based on these tensile test results, we can suggest that, among all of the samples, the EVA nanocomposite with 1 wt% MMT is the most biostable material. When the nanofiller content was increased to 3 and 5 wt%, the biostability decreased as a result of increased organo-MMT agglomeration and larger tactoids.

Table 3 The percentage increase in mass of EVA and EVA nanocomposites after the in vitro exposure to PBS solution at 37 °C

Material	Increase in mass upon 4 weeks of in vitro exposure (%)
EVA	0.14
EVA + 1 wt% MMT	0.08
EVA + 3 wt% MMT	0.18
EVA + 5 wt% MMT	0.34

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3.5. Water permeability (exposure to the PBS solution at 37 °C)

It is well understood that the factors that can affect the permeability of the polymer nanocomposite to liquid water include the nanofiller loading, degree of dispersion, defects at the polymer–filler interface, misalignment of platelets, disrupted molecular packing and increase in the size of free volume elements in the polymer, which can result in an increased rate of water transmission through the polymer.^33–35 In this study, a water permeability test was performed to investigate the hydration characteristics of the EVA and EVA nanocomposites and the relationship of these characteristics with the mechanical properties. The analysis was done by immersing the samples in the PBS solution at 37 °C. The results are summarized in Table 4 and Fig. 5. It is observed that the water uptake was highest in the nanocomposites with 5% organo-MMT, upon 4 weeks of hydrolytic exposure. This is based on the percentage increase in mass (0.34%), which was found to be higher than that of neat EVA. However, EVA exhibits a reduction in water uptake when 1% organo-MMT is incorporated. This nanocomposite displays the lowest percentage (0.08%) of mass increase as compared to the other materials. These results are in agreement with the TEM analysis, which indicated that the best nanofiller dispersion characteristics in the EVA matrix were obtained with the sample containing 1 wt% organo-MMT. The well dispersed and exfoliated hydrophobic nanoplatelets may create a more tortuous path for the water molecules’ diffusion, and restrict their entrance into the EVA copolymer chains.

Table 4 Tensile properties of neat EVA and EVA nanocomposites before and after 4 weeks of in vitro exposure to PBS solution at 37 °C

Mechanical properties	PBS exposure	EVA	EVA + 1 wt% MMT	EVA + 3 wt% MMT	EVA + 5 wt% MMT
Tensile strength (MPa)	Before	12.3 ± 0.7	14.3 ± 0.6	13.0 ± 0.4	12.6 ± 0.1
	After	11.6 ± 2.0	13.8 ± 1.1	12.2 ± 0.6	12.0 ± 1.3
Elongation at break (%)	Before	624 ± 26	686 ± 24	647 ± 15	664 ± 32
	After	768 ± 32	875 ± 20	812 ± 36	808 ± 44
Toughness (MPa)	Before	43.9 ± 3.3	55.7 ± 3.0	49.7 ± 1.0	49.9 ± 1.8
	After	51.2 ± 1.6	66.2 ± 4.9	55.4 ± 4.9	54.7 ± 8.6

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Fig. 5 Representative stress–strain curves for neat EVA and EVA nanocomposites before and after 4 weeks of in vitro exposure to PBS solution at 37 °C.

3.6. Ambient and in vitro mechanical properties after immersion in PBS solution at 37 °C

The tensile properties of the neat EVA and EVA nanocomposites before and after exposure to a hydrolytic agent, PBS, at 37 °C are summarized in Tables 4 and 5, and the representative stress–strain curves are shown in Fig. 5. As expected, the nanocomposite incorporating 1 wt% MMT exhibits the best in vitro mechanical properties, due to its enhanced water impermeability. This can also be associated with the presence of well dispersed and exfoliated organo-MMT layers. Interestingly, the effect of the hydrolytic agent on the mechanical properties was different to that of the oxidative agent. It was found that the tensile strength was decreased for all of the samples, but the elongation at break and toughness were increased when they were in their hydrated states.

Table 5 Reduction in tensile strength and toughness of EVA and EVA nanocomposites after 4 weeks of in vitro exposure to PBS at 37 °C

Materials	% reduction in tensile strength after PBS exposure	% increase in toughness after PBS exposure
EVA	5.0	14.1
EVA + 1 wt% MMT	3.4	15.9
EVA + 3 wt% MMT	6.6	10.2
EVA + 5 wt% MMT	4.9	8.6

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The reduction in the tensile strength of the hydrated EVA is expected, as the water molecules can occupy the hydrogen bonding sites and subsequently hinder the secondary bonding between the copolymer chains. The accompanied increase in the elongation at break and toughness can probably be attributed to an increase in the elasticity of EVA due to the plasticization of vinyl acetate upon water inclusion. Water molecules may be more attracted to the polar vinyl acetate structure, penetrate into the molecular chains and plasticize them.³⁶ It was hypothesized that these interactions contribute to the enhanced elasticity and toughness of EVA at 37 °C, as they promote a higher degree of conformation and relaxation at local stress regions compared to the ambient conditions (with a lower temperature). The toughening effect is somehow more pronounced in the EVA containing the organo-MMT (Fig. 5). This suggests that the chain segment mobility is further enhanced by the presence of the organic onium ions from the organo-MMT, especially when less water is absorbed into the structure. This is because the onium ions can provide greater binding energy with the polyethylene chains, and this interaction results in enhancement in molecular dynamics and conformation freedom at the clay platelets–matrix interface. One could possibly question why the stress–strain behaviour of EVA has been moderately altered by the inclusion of the nanofiller. The explanation that we have provided is illustrated in Fig. 6. At low strain, the presence of tactoids may introduce interference in the load transfer efficiency, thus preventing the increase in Young’s modulus. However, when high strain is reached, the organo-MMT becomes exfoliated, which results in a greater number of single nanoplatelets, which are more preferentially aligned in the direction of the stress and introduce a more tortuous path for the diffusion of the permeant (water). We postulate that these are the reasons for the enhancement in the ultimate strength, toughness and biostability of EVA, which are shown in the EVA nanocomposite with 1 wt% organo-MMT. Apparently, these are molecular interactions that would be very interesting to explore further, therefore future studies will be performed to elucidate their nature in greater detail.

Fig. 6 Schematic showing the morphology of the EVA nanocomposites at low and high strains.

The best retention in tensile strength and toughness upon PBS solution exposure was achieved when 1 wt% organo-MMT was added to EVA. The reduction in tensile strength was only 3.4%, as compared to 5.0% for the neat EVA. This is in line with the water permeability data, which suggest that this material absorbs less water upon 4 weeks of exposure to a hydrolytic agent.

3.7. Biocompatibility assessment

The biocompatibility of the neat EVA and EVA nanocomposites was assessed by observing the attachment and proliferation of Chang liver cells on the surface of these materials. The attachment and growth of the cells on the surface of the polymers gives an initial and important indication of their biocompatibility. In the absence of cytotoxic substances, the cells may undergo further development, such as differentiation and proliferation processes. Based on SEM analysis (Fig. 7), we suggest that, with the applied incubation time and conditions, the EVA and EVA nanocomposites incorporating organo-MMT do not produce leachable substances that contribute to the inhibition of the Chang liver cells’ growth. The similar cell morphology observed upon the incubation of the cells with the neat EVA and EVA nanocomposites implies that the biocompatibility of the EVA incorporating the organo-MMT is similar to that of neat EVA. Cells were tightly attached to the surface of the polymers even after 96 hours of incubation time, indicating that no harmful substances were released that were toxic to the liver cells. Observation of the Chang liver cells using scanning electron microscopy, however, indicated that cell adhesion was slightly favoured in the EVA incorporating 1 wt% organo-MMT as compared to the neat EVA and other nanocomposites (see Fig. 7). It was observed that the filopodia of the Chang liver cells attached onto the polymer surface were thicker than those of the cells attached onto the other polymers with a higher percentage of organo-MMT. This might be attributed to the dispersion of the organoclay filler and its interaction with the host EVA, which resulted in the smoother and homogeneous surface morphology of the EVA + 1 wt% organo-MMT nanocomposite sample (Fig. 2 and 3), thereby allowing more favourable filopodia growth and attachment. Previous research also proved that the biocompatibility of the host polymer is greater if the dispersion and exfoliation of the organoclay filler is better.⁵ It is also noticeable that the cells incubated with the neat EVA sample resulted in a thin and short filopodia structure extending from the cell cytoplasm, and the cells became rounded in form. The enhancement of the filopodia attachment with the addition of 1 wt% organo-MMT into the host EVA can be hypothesized to result from cell adherence to both the EVA and organo-MMT structures. However, the exact mechanism for this phenomenon has yet to be elucidated.

Fig. 7 Scanning electron micrographs of Chang liver cells grown on a glass cover slip (a), EVA + 1 wt% MMT (b), EVA + 3 wt% MMT (c), EVA + 5 wt% MMT (d) and neat EVA (e) after 96 hours of incubation time. Cells are seen to attach onto the glass cover slip (control) and polymer surfaces. Arrows indicate the filopodia structures of the Chang liver cells.

Having benchmarked the morphologies of the cells attached to the nanocomposites against those of cells attached onto the surface of the control medium (glass cover slip), we can suggest that the EVA nanocomposites do not inhibit the growth and proliferation of cells even after 96 hours of incubation on their surfaces. The ability of the cells to remain attached onto the polymer surface is due to the biocompatibility of the polymers towards the Chang liver cells and it can be concluded that the organo-MMT nanofiller used in this study did not cause hepatotoxicity during this biosafety assessment.

4. Conclusions

The concentration of the organo-MMT might affect the arrangement of the MMT nanoplatelets, and thus also their exfoliation and intercalation behaviour in the EVA matrix. It was noticeable that the dispersed organo-MMT (added in the amounts of 1, 3 and 5 wt%) have a few large stacks (tactoids) that still remain throughout the EVA matrix. This suggests that the shear energy provided by the Brabender mixer was not sufficient to break and peel some of the large tactoids into individual clay layers. The degree of MMT dispersion and exfoliation decreased as the nanofiller loading increased. As revealed by both XRD and TEM analysis, the EVA nanocomposite containing 1 wt% MMT had the greatest degree of nanofiller dispersion and exfoliation among all of the nanocomposites. The SEM images illustrated signs of surface degradation in the neat EVA and EVA nanocomposites upon 4 weeks of oxidative exposure. The surface degradation features of the EVA nanocomposites containing 1 wt% MMT were seen to be smoother than those of neat EVA and other nanocomposites after four weeks of oxidative treatment. This can be related to the morphology of this material, which shows better dispersed and exfoliated organo-MMT distributed throughout the EVA matrix. A slower degradation process took place, as the diffusion of the oxidant molecules was restricted by a greater number of nanoplatelet layers. The tensile properties of the neat EVA and EVA nanocomposites (before and after exposure to an oxidative and hydrolytic agent) were studied. The best mechanical properties were exhibited by the nanocomposite with 1 wt% organo-MMT, which was also less affected by the oxidative and hydrolytic treatments than the other materials. The superior in vitro mechanical properties of the EVA nanocomposite with 1 wt% MMT can be associated with its morphology, as indicated by XRD, TEM, SEM and also its permeability characteristics. We hypothesized that the well dispersed and exfoliated organo-MMT (MMT whose surface was modified with a hydrophobic surfactant) created a more tortuous path for the diffusion of oxidants and water molecules, thereby hindering more severe degradation in the EVA molecular chains. In addition to its surface degradation features, the superior mechanical properties of this particular material have provided further evidence of its enhanced biostability. The results also demonstrate that hydrated EVA (which was exposed to PBS at 37 °C) was further toughened upon the addition of 1 wt% organo-MMT. The outcome of the biocompatibility test employing the co-cultivation of Chang liver cells suggests that the EVA nanocomposites did not cause hepatotoxicity when incubated with the cells. The incorporation of 1 wt% of organo-MMT into the host EVA resulted in the enhancement of filopodia growth and attachment on the surface of this copolymer. These preliminary studies revealed that the EVA–organo-MMT nanocomposites have the potential to be further developed for biomedical applications, thus more research and development of these materials should be carried out.

Acknowledgements

This research was funded by Fundamental Research Grant Scheme (FRGS) (9003-00473) from the Ministry of Education, Malaysia. The authors thank Mr Omar S. Dahham, Mr Mohd Syahmi Mohd Rasidi and Mrs Noormarlyna Ismail for technical help during the melt compounding and testing processes.

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