PLA-ZnO nanocomposite films: Water vapor barrier properties and specific end-use characteristics

Accepted Manuscript PLA-ZnO nanocomposite films: water vapor barrier properties and specific enduse characteristics Roberto Pantani, Giuliana Gorrasi, Giovanni Vigliotta, Marius Murariu, Philippe Dubois PII: DOI: Reference:

S0014-3057(13)00414-X http://dx.doi.org/10.1016/j.eurpolymj.2013.08.005 EPJ 6196

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

30 December 2012 30 July 2013 4 August 2013

Please cite this article as: Pantani, R., Gorrasi, G., Vigliotta, G., Murariu, M., Dubois, P., PLA-ZnO nanocomposite films: water vapor barrier properties and specific end-use characteristics, European Polymer Journal (2013), doi: http://dx.doi.org/10.1016/j.eurpolymj.2013.08.005

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PLA-ZnO nanocomposite films: water vapor barrier properties and specific end-use characteristics Roberto Pantani[a]*, Giuliana Gorrasi[a], Giovanni Vigliotta[b], Marius Murariu[c] and Philippe Dubois[c] [a] Department of Industrial Engineering, University of Salerno, via Giovanni Paolo II, 84084 Fisciano (SA)-Italy [b] Department of Chemistry and Biology University of Salerno, via Giovanni Paolo II, 84084 Fisciano (SA)-Italy [c] Center of Innovation and Research in Materials & Polymers (CIRMAP), Laboratory of Polymeric and Composite Materials (LPCM), University of Mons & Materia Nova Research Centre, Place du Parc 20, 7000 Mons, Belgium * e-mail: [email protected]

PLA nanocomposite films with multifunctional characteristics such as mechanical, anti-UV, antibacterial, electrical, gas barrier properties are potentially of high interest as packaging biomaterials. Occasionally, desired and beneficial effects obtained by addition nanofillers come along with some drawbacks, leading to the sharp drop in the molecular weights of the polyester chains, and consequently an important loss of mechanical and thermal properties. Novel PLAZnO nanocomposite films were produced by melt-compounding PLA with 0.5-3% ZnO rod-like nanoparticles. The surface treatment of nanofiller by silanization (with triethoxy caprylylsilane) was necessary to obtain a better dispersion and to limit the decrease of molecular mass of PLA. The morphology, molecular, thermo-mechanical and transport properties to water vapor of PLAZnO films were analyzed with respect to the neat PLA. According to DSC and to XRD, the

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produced films were essentially amorphous. The changes in PLA permeation properties were strongly dependent on temperature and nanofiller loading. The well dispersed ZnO nanoparticles within the polyester matrix were effective in increasing the tortuosity of the diffusive path of the penetrant molecules. The activation energy remained similar for PLA and PLA-1% ZnO, but was found greater at higher loading of ZnO (3%), confirming the increased difficulty of travelling molecules to diffuse through PLA. In comparison to the neat PLA (presenting no antimicrobial efficacy), the nanocomposites were active against both Gram positive and Gram negative bacteria, stronger antibacterial activity being evidenced after 7 days elapsed time. By considering the multifunctional properties of PLA-ZnO nanocomposites, the films produced by extrusion can be considered a promising alternative as environmental-friendly packaging materials.

Keywords: Poly(lactic acid); nanocomposite films; zinc oxide; packaging; films; water vapor permeability.

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1. Introduction The need to extend the shelf life of packaged food has brought the research to innovative solutions along with the changing needs of consumers, making the "packaging" a constantly evolving field [1-3]. The current trend is directing the research towards the development of innovative solutions both for functional packaging (active packaging and nanocomposite materials) and low environmental impact (biodegradable materials, recyclable packaging with reduced size). Most films, used to preserve food stuff, have been produced from synthetic polymers. Nevertheless, for environmental reasons, attention has lately been focused on biodegradable polymers for the preparation of food packaging films [4–13]. These films are usually loaded with antimicrobial agents that come into contact with food stuff, act on food-born microorganisms and inhibit their growth [14-21]. Therefore, the current research has been focused on the search for new bactericides that can effectively reduce the harmful effects of microorganisms. With the emergence of nanotechnology, the search for effective biocidal agents has focused on the development of nanostructure of coinage metals like silver, copper, zinc and gold [22]. However, the high cost of silver and gold metals has limited their use as antibacterial agents on industrial basis. Therefore, currently metal oxide nanoparticles, such as ZnO, have emerged out as a new class of important materials that are increasingly being developed for use in research and health-related applications, because of their low cost, easy availability, and unique chemical and physical properties. Recently, several reports have described the antimicrobial activity of ZnO nanoparticles [23-26]. Polylactide (PLA) is an environmental friendly, economical and commercially available polymer that offers great potential as disposable packaging material [27-31].

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ZnO nanoparticles are well-known environmentally friendly and multifunctional inorganic additives that could be considered as nanofillers for various polymers providing properties like antibacterial effect or intensive ultraviolet absorption [32, 33]. In this context, it is certainly of interest to endow PLA with antibacterial properties (and intensive ultraviolet absorption) and addition of ZnO nanoparticles to PLA could represent a relevant approach [34-36]. Unfortunately, the addition of untreated ZnO nanoparticles into PLA at melt-processing temperature leads to severe degradation of the polyester matrix, ascribed to the transesterification reactions and ‘unzipping’ depolymerization of PLA. By contrary, as reported elsewhere [34], ZnO adequately surface-treated by selected silanes can lead to PLA-based nanocomposites characterized by quite good preservation of PLA intrinsic molecular parameters and related physicochemical characteristics. In this paper we report the preparation of PLA-ZnO nanocomposites in a composition range from 0.5 to 3% nanofiller, followed by the extrusion of films. This is one of the first studies concerning the main characteristics of PLA-ZnO nanocomposite films, with a special focus on the water vapor barrier properties resulted from nanofiller incorporation. The morphology of the so-produced films and their thermal, mechanical and transport properties (sorption (S), diffusion (D), permeability (P)) to water vapor were analyzed. The investigation of transport properties was conducted at three different temperatures to evaluate also the activation energy of the diffusion phenomenon. In order to test the used ZnO as antimicrobial agent in the prepared film nanocomposites, two different types of bacteria (Gram-positive and Gram-negative) were used to evaluate the degree of inhibition of bacterial growth depending on the incubation time and filler amount.

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2. Experimental 2.1 Materials Poly(L,L-lactide) – hereafter called PLA, supplier NatureWorks LLC, was a grade designed for realization of films (4032D) with Mn= 133 000, dispersity, Mw/Mn = 1.9, whereas according to producer information the other characteristics are as follows: D isomer = 1.4%; relative viscosity = 3.94; residual monomer = 0.14%. Commercially available ZnO nanofillers (rod-like particles) were kindly supplied by Umicore Zinc Chemicals (Belgium) as Zano 20 Plus (surface coated with a silane especially suitable for the treatment of metal oxides, i.e., triethoxy caprylylsilane; ZnO content: 96.2 ± 0.5%, bulk density: 360 g/L). A thermal stabilizer, Ultranox 626A, (bis (2,4-di-t-butylphenyl) pentaerythritol diphosphite) was used at 0.3% in PLA. Throughout this contribution, all percentages are given as wt %. 2.2 Preparation of nanocomposites and films PLA-ZnO nanocomposites were produced by melt compounding PLA with up to 3% ZnO nanofiller in a Leistritz twin-screw extruder (type ZSE 18 HP-40D, diameter of screws (D) = 18 mm, L/D = 40). The previously dried granules of PLA and additives were first mixed in a Rondol turbo-mixer (2000 rpm, 2 min) with 0.5, 1, 2 and 3% ZnO, followed by dosing and melt compounding in twin-screw extruder (throughput of 1.5 kg/h, speed of the screws = 100 rpm, temperature of the molten polymer ~185 °C). For the sake of comparison, unfilled PLA containing only thermal stabilizer was processed in similar conditions of melt-compounding. The granules of unfilled PLA and PLA-ZnO nanocomposites were dried (at 80 °C overnight, under vacuum) and used for the production of films by extrusion. Films with a thickness of about 150 µm were obtained using a DSM twin-screw microcompounder (batch-volume: 15 mL, speed of

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screws: 70 rpm, temperature of molten polymer: 185-190 °C) equipped with a flat die (width: 35 mm, die opening: 0.4 mm) and a DSM Xplore microfilm device. 2.3 Methods of characterization Molecular weight parameters (number average molar mass, Mn, and dispersity index, Mw/Mn) of unfilled PLA and PLA-ZnO films were determined by size exclusion chromatography (SEC). Recovery of PLA from selected compositions for molecular weight parameters determination was carried out by firstly dissolving the samples in chloroform. The metallic residues were removed by liquid–liquid extraction with a 0.1N HCl aqueous solution, step followed by intensively washing with demineralized water. Finally, PLA was recovered by precipitation in an excess of heptane. After filtration and drying, PLA solutions were prepared in chloroform (10 mg polymer / 5 ml solvent). Molecular weight parameters of pristine PLA and those of PLA extracted from the studied nanocomposites were determined by SEC after a previously filtration of PLA solutions using filters of 0.45 µm. X-ray diffraction measurements (XRD) were performed with a Bruker diffractometer (equipped with a continuous scan attachment and a proportional counter) with Ni-filtered Cu Kα radiation (λ = 1.54050 Å). Differential scanning calorimetry (DSC) analysis was carried out on samples with a mass ranging between 8 and 12 mg. The tests were carried out by means of a DTA Mettler Toledo (DSC 30) under nitrogen atmosphere. The samples were heated from -60 °C to 250 °C at 10 °C/min. To ensure reliability of the data, heat flow and temperature were calibrated with standard materials, i.e., indium and zinc. The events of interest, i.e., the glass transition temperature (Tg) and the associated enthalpy of relaxation (∆Hrel), cold crystallization temperature (Tc), enthalpy of cold crystallization (∆Hc), melting temperature (Tm) and melting enthalpy (∆Hm), were

6

evaluated. The degree of crystallinity (χc) was determined by subtracting ∆Hc from ∆Hm, and by considering a melting enthalpy of 93 J/g for 100% crystalline PLA [34]. Barrier properties (sorption, diffusion and permeability) were evaluated using a microbalance SMS DVS Advantage-2 system. This system has a sensitivity of ±1.0µg, and allows the measurements of mass changes due to sorption or desorption of vapor molecules. In this work the chosen method consisted in submitting the sample to pressure steps at constant temperature. The tests were conducted using water vapor in a nitrogen atmosphere at 30 °C. The starting samples were dry, square films having a thickness of 150µm and a side of 15mm. The experimental protocol considered steps of relative humidity from 0 to 80%. The chosen temperatures were: 15; 30 and 45 °C. Tensile testing measurements were performed using a Lloyd LR 10K tensile bench on strips cut from films (63.5 x 10 x ~0.15 mm3) at a speed rate of 0.5 mm/min, with the distance of 40 mm between grips. All tests were carried out on specimens previously conditioned for at least 48 hours at 20 (±2) °C under a relative humidity of 50 (±3) % and the values were averaged out over minimum five measurements from each sample. Opacity measurements were performed using a Konica Minolta CM-2500d X-Rite SP60 Series spectrophotometer. Following the ASTM E284 (“Terminology of Appearance”), the opacity was defined as ability of a thin film to hide a surface behind and in contact with it, expressed as the ratio of the reflectance factor (Rb) when the material is backed by a black surface to the reflectance factor (Rw) when it is backed by a white surface (usually having a reflectance factor of 0.89). The opacity (O) was calculated using the relationship: O (%) = (Rb/Rw) x 100

1

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In order to evaluate the antibacterial effect at different ZnO loading, Escherichia coli and Staphylococcus aureus were pre-inoculated in Luria-Bertani (LB) medium (10 gL-1 trypton, 5 gL-1 yeast extract, 10 gL-1 NaCl) at 37 °C and grown for 12 h in aerobiosis at 250 rpm. Subsequently, bacteria were collected by centrifugation for 10 min at 3500 g, re-suspended at concentration of 0.005 OD600 in presence of 1 cm2 of polymeric films, each cut into four equal parts, and incubated in aerobiosis to 37 °C at 250 rpm. E. coli more resistant to oligotrophic conditions was re-suspended in distilled, sterile water, while S. aureus more sensitive to lack of nutrients was re-suspended in diluted (50% vol/vol) peptone water (1 gL-1 peptone, 8.5 gL-1 NaCl). For cell survival determination at indicated times 50 µl of different dilutions of each suspension were spread on LB agar dishes (15 gL-1 agar), incubated for 24 hours and colony forming units (CFU) calculated. The obtained values were used to calculate the antibacterial activity (A) as previously reported [34]. Briefly, was applied the formula: A=F–G

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F represents the growth values in presence of unfilled PLA samples (control/without ZnO), while G corresponds to the growth values of filled samples (PLA-ZnO films). They are calculated according to the formula F = Log (C24h/7d - Log C0) and G = Log (T24h/7d - Log T0). C and T are CFU detected respectively for control and filled nanocomposite material at different times, i.e., 0, 24 hours and 7 days. Antibacterial efficacy was tested on films with 1, 2 and 3% ZnO. The PLA-ZnO films were considered “antimicrobial” when achieving an A superior to 2 (reduction in bacteria number > 99 %). Transmission electron microscopy (TEM): TEM images of nanofiller and selected PLA-ZnO nanocomposites were obtained with a Philips CM200 apparatus using an accelerator voltage of

8

up to 120 kV. The nanocomposite samples (70–80 nm thick) were prepared with a Leica Ultracut UCT ultracryomicrotome by cutting at -100 °C.

3. Results and discussion

Preliminary considerations In order to make PLA matrix less susceptible to the catalytic action of ZnO nanoparticles during the melt blending process and subsequent film processing, the nanofiller surface treatment with silane agents was considered as a prerequisite, leading to significant enhancements of both thermo-mechanical and molecular properties with respect to the use of untreated ZnO [34]. In relation to the transport properties of PLA films, it is noteworthy to mention that according to H. Tsuji et al. [37], the changes in Mn of PLLA films in the range of 9 104 - 5 105 g mol-1and Dlactide unit content up to 50%, lead to insignificant effects on the water vapor transmission rate (WVTR). In contrast, the WVTR of PLA films were found to decrease monotonically with increasing the degree of crystallinity from 0 to 20%, while levelling off for values exceeding 30%. Table 1 reports the modification of PLA molecular characteristics upon films production. PLA is very sensitive to hydrolysis, shearing and to the contact with Zn-based products. The molecular characterization of unfilled PLA and PLA-ZnO nanocomposite films confirms the expected decreasing of Mn and changes of dispersity index after the melt-compounding and following the second processing to produce films by extrusion. The Mn of PLA was about 101 000Da, whereas the films containing 1 and 3% ZnO were characterized by a Mn of 78 500Da and 68 200Da, respectively. Accordingly, a noticeable

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decrease of Mn (about 32 000Da) and significant change of dispersity index are seen even for the neat PLA film. However, as previously reported [34], once more it is confirmed that the addition of silane treated ZnO does not lead to a dramatic drop of PLA molecular weights, which was not the case of untreated nanofiller. Based on previous data reported in the literature [37], for an easier interpretation of the results we will assume that the barrier proprieties of the films are marginally influenced by the molecular parameters, whereas the information concerning the level of crystallinity is presented in the next section.

Table 1. Determination of molecular weights and dispersity index of neat PLA and PLA-ZnO films Entry Sample (%, by weight) Dispersity index Mn [Da] 1

PLA (granules)

1.9

133 000

2

PLA (0% ZnO)

2.6

101 200

3

PLA- 1% ZnO

2.6

78 500

4

PLA- 3% ZnO

2.9

68 200

Morphology and crystallinity In the case of the studied nanocomposite films, the system contains a continuous phase represented by the PLA matrix and a dispersed phase, the ZnO nanofiller. Following the TEM investigations (Fig. 1a) the nanoparticles are characterized by a rod-like morphology, typically with diameters of ~15-30 nm and a length up to around 100 nm. Mineral surfaces covered with hydroxyl groups such as ZnO are generally very receptive to the bonding with alkoxysilanes and more than one (mono) layer of silane is usually applied onto the surface of the filler, which could

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play the role of compatibilizer and dispersion agent. In the nanocomposites with 1% naanofiller the ZnO nanoparticles are quite well distributed and dispersed through the PLA matrix (Fig. 1b and c), since the presence of aggregates is difficult to be observed in the TEM pictures. Furthermore, by increasing the loading of ZnO at 3% the distribution of the nanofiller is remaining reasonably good (Fig. 1d), whereas the TEM images realized at higher magnification (Fig. 1e) are proving that are only few zones where some ZnO nanorods are associated as small clusters. On the other hand, even though the loading (up to 3%) and consequently the volume fraction of ZnO nanofiller is not so high in nanocomposite films, it is believed that the nanoparticles can provoke a tortuosity to water vapor, pure or mixed gas, such in the case of other (nano)composites, finally leading to changes of PLA inherent permeation properties. [38-40]

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Figure 1 (a – e). TEM images at different magnifications to illustrate the morphology of ZnO nanoparticles (a) and those of PLA nanocomposites filled with 1% ZnO (b and c) and 3% ZnO (d and e)

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Furthermore, in semi-crystalline polymers, the crystalline regions are considered to be impermeable to the vapor molecules, and in this context it is of interest to have information about the morphology and structures of initial samples as they are evaluated by the different techniques. However, by considering the specific experimental procedure, it is assumed that the PLA crystallinity of prepared films could be mainly influenced by the molecular characteristics, the content of D-isomer, the parameters of processing by extrusion (residence time, shear, drawing and cooling to produce the films), etc. The quantification of PLA crystallinity on films was carried out mainly by DSC whereas XRD was used as additional tool of investigation. Table 2. Calorimetric data from DSC thermograms of PLA and PLA-ZnO films Tg

∆Hrel

Tc

∆Hc

Tm

∆Hm

χc

(°C)

(J/g)

(°C)

(J/g)

(°C)

(J/g)

(%)

PLA

64

5.8

108

26.4

169

32.5

6.6

PLA- 0.5% ZnO

65

5.6

110

32.2

170

35.0

3.0

PLA- 1% ZnO

64

6.1

111

34.2

171

36.8

2.8

PLA- 2% ZnO

64

4.7

110

32.3

171

35.1

3.0

PLA- 3% ZnO

63

5.5

111

34.1

172

36.7

2.8

Sample

Figure 2 shows the DSC thermograms of all nanocomposites and of neat PLA, whereas the calorimetric parameters are summarized in Table 2. All samples are characterized by a marked exothermic crystallization as confirmation of the amorphous or low crystalline starting structures. The glass transition temperature (Tg) and the endothermic enthalpy of relaxation (∆Hrel) do not show significant modifications with nanofiller addition during the first DSC scan. This phenomenon of enthalpic relaxation is typical for a polymeric material in the glassy state that undergoes physical ageing [28, 41]. The samples display a similar cold crystallization 13

temperature (Tc), whereas the nanocomposites show higher enthalpy of cold crystallization (∆Hc) with respect to neat PLA. However, it is of interest to note that all nanocomposite films were characterized by a very low degree of crystallinity (χc), i.e., about 3%, while neat PLA showed a slightly higher value, i.e., 6.5%. Accordingly, the analyzed films are largely characterized by an amorphous structure, results that are not surprising by considering the poor crystallization ability of PLA [42, 43]. Furthermore, it is assumed that the eventual orientations and rearrangements of macromolecular chains into a crystalline structure under the specific extrusion conditions are rather negligible. Following the DSC analysis, it is evident that the addition of up to 3% ZnO (thus surface-treated with silane agents), does not lead in this typical experiment to an increase of PLA crystallinity, a conclusion that is also supported by the results of XRD investigations.

Figure 2. DSC thermograms of neat PLA and all PLA-ZnO nanocomposites. The curves are arbitrarily shifted along the vertical axis

Figure 3 shows the XRD patterns of neat PLA and all studied nanocomposite films. On one hand, all samples show a broad intensity with a maximum appearing at approximately 2θ = 17°

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indicating mainly an amorphous structure [44]. Furthermore, according to the XRD measurements and consistent with the DSC results, the samples filled with 1 - 3% ZnO are completely amorphous or only slightly crystalline. On the other hand, due to some traces of crystallinity, only the neat PLA and the nanocomposites film containing the lower amount of ZnO (0.5%) exhibit an evident crystallization peak (close to 2θ of ~ 16.5°) ascribed to the diffraction of (200) and/or (110) planes of the typical orthorhombic crystal of PLA. [45]. Furthermore, the specific peaks evidenced at 2θ= 31.6° and 2θ= 36.2° can be ascribed respectively to the diffraction planes of (100) and (101) of the crystalline form of ZnO, while their intensity is clearly increasing with nanofiller loading [46]. Finally, because the investigated PLA samples were mainly amorphous, in the discussion of results it was considered that the crystallinity is not an additional parameter that can influence the transport properties.

Figure 3. X-ray diffraction patterns for PLA and all PLA-ZnO nanocomposites

Barrier properties It is well known that biodegradable polyesters, like PLA, are moisture sensitive [47-48], so the analysis of transport properties (sorption, diffusion and permeability) to water vapor is of 15

fundamental importance for developing new packaging materials. Water molecules, at different activity and temperatures, could have effect on the microbial growth and influence the shelf life of the packed products. On the other hand, the presence of filler could influence the transport phenomena of small molecules through a polymeric matrix either from a thermodynamic or a kinetic viewpoint. For this purpose, we performed the analysis of the thermodynamic parameter, sorption (S), the kinetic parameter, diffusion (D), and their product, the permeability (P = S x D) at three different temperatures (i.e., 15, 30 and 45 °C) on unfilled PLA and PLA filled with 1% and 3% ZnO.

Table 3. Sorption parameters, S (wt%/atm), evaluated from isotherms of Fig. 4 using Eq. 3 Sample → PLA Temperature , °C↓

PLA-

PLA-

1% ZnO

3% ZnO

15

44.90

46.15

46.05

30

20.58

20.50

20.22

45

9.94

10.00

9.98

Sorption Some modifications of polymers by swelling, hydrolysis or plasticization can result from the sorption of water, which can be accelerated by the presence of polar and hydrophilic groups. [47] Sorption isotherms were obtained for PLA and nanocomposites loaded with 1% and 3% ZnO. Figure 4 reports the equilibrium concentration of water vapor, Ceq (wt%), as function of water partial pressure, P (atm) at the investigated temperatures for unfilled PLA and PLA-ZnO nanocomposite films. All samples show an ideal behaviour at low pressure, that allowed evaluating the sorption coefficient, S, from Henry’s law: 16

Ceq= S x P

3

For isotherms at 15 and 30 °C we considered the first four points, very well interpolated from Henry’s model. In the case of isotherms evaluated at 45 °C, for the evaluation of sorption, we have taken into account the first three points, which ensure an ideal interaction matrix-penetrant. It is evident that, in all cases, at high pressure the isotherms deviate from linearity following a Flory-Huggins mode of sorption [48] (see the arrows in the plots). According to this behaviour there is preference for the formation of penetrant-penetrant pairs, so that the solubility coefficient continuously increases with penetrant pressure. The first molecules sorbed tend to locally loose the polymer structure and make it easier for the following molecules to enter. These isotherms are observed when the penetrant effectively plasticizes the polymer, being a strong solvent or swelling agent for the polymer, like is water vapour for PLA matrix. Table 3 reports the S (wt%/atm) parameters, evaluated according to Eq. 3, for the analyzed samples at the three studied temperatures. At constant temperature, there is not evident variation of S with increasing ZnO loading. Comparing the data evaluated at different temperatures, it is possible to observe a decreasing of sorption with increasing the temperature, in agreement with the thermodynamics of the system.

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Figure 4. Ceq of water vapor (wt%) vs. the partial pressure of water vapour at different temperatures for PLA, PLA- 1% ZnO and PLA- 3% ZnO.

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Table 4: Thermodynamic diffusion coefficients, Do (cm2/s), at different temperatures (15; 30, 45 °C) and diffusion activation energy, ED (kJ/mol) for PLA and nanocomposite films loaded with 1% and 3% ZnO. Do and ED values should be considered as having an accuracy of about ±10%. Sample

Temperature, °C

PLA

PLA- 1% ZnO

PLA- 3% ZnO

Diffusion Coefficient, Do (cm2/s)

15

3.27 10-8

30

7.75 10-8

40

10.4 10-8

15

3.75 10-8

30

5.71 10-8

40

11.7 10-8

15

2.51 10-8

30

4.85 10-8

40

11.1 10-8

Diffusion Activation Energy (ED), kJ/mol

29.60

28.73

37.63

Diffusion Following the increasing of sample weight as function of time, it was possible to evaluate the diffusion coefficient, D, at different water pressures [49]. The equation used to evaluate the diffusion parameter at different water vapor partial pressures is as follows: 4

in which M(t) is the mass of the sample at each time, M0 is the value at the beginning of the test, M∞ is the value at equilibrium, h is the thickness of the sample. Eq. 4 is valid for times for which the ratio [(M-M0)/(M∞-M0)] is larger than 0.4. Plotting ln(d Mt/dt) vs. time, the value of D

19

(cm2/s) is calculated at each partial pressure from the slope of the curve. Figure 5 shows an example of the procedure adopted to determine the diffusivity. On the left axis it is possible to read measurements of the normalized mass of PLA at 30 °C (step of humidity from 0 to 20%). The squares show the calculations performed according to Eq. 4. The range of points to fit was always determined by a mass change between 40% and 90%. Accordingly, for a time long enough the considered model fits very well the experimental data. By the knowledge of sample thickness, the diffusion coefficient is evaluated from the slope of the line.

Figure 5. Example to illustrate the procedure adopted to determine the diffusion coefficients.

The diffusion parameters obtained using Eq. 4 were plotted as function of water percentage sorbed at the different pressures. For polymer-solvent systems, the diffusion parameter is usually not constant, but depends on the vapor concentration, according to the empirical Eq. 5: D = Do exp (γ Ceq)

5

where Do (cm2/s) is the zero concentration diffusion coefficient (related to the fractional free volume and to the microstructure of the polymer); γ is a coefficient which depends on the fractional free volume and on the effectiveness of the penetrant to plasticize the matrix.

20

Figure 6 reports the diffusion coefficient, D (cm2/s), as function of the equilibrium concentration of water vapor, Ceq (wt%), for unfilled PLA and nanocomposite films (1 and 3% ZnO) at 15, 30 and 45 °C. It is evident that the diffusion is independent of water vapor concentration at any investigated temperature, so that the Do values extrapolated at Ceq= 0, with very good degree of approximation can be considered equal to D at any vapor pressure. Do values are reported in Table 4. As expected, on increasing the temperature, Do increases. Therefore, the diffusion process is faster by increasing polymer free volume and at higher mobility of polymeric chains. At constant temperature a slight decrease of D with ZnO loading can be observed, more evident at T= 30 °C. A great number of data in literature suggest that the diffusion coefficient of vapors in polymers depends on temperature, via an Arrhenius’ law on a narrow range of temperatures following the Eq. 6: 6 where Do represents the limit value of the transport coefficient for T (temperature) →∞ and ED (kJ/mol) is the diffusion activation energy, i.e., the energy level that a molecule must reach to diffuse inside the polymeric matrix. In our case, D being constant with Ceq, it was possible to plot Do vs. 1/T (T in K) to evaluate the activation energies (Figure 7) that are reported in Table 4. It is evident that such values remain quite constant for PLA and PLA- 1% ZnO, but are higher for the sample containing 3% ZnO, confirming the increased difficulty of the travelling molecules to diffuse into the polyester matrix.

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1.00E-6

PLA+3% ZnO 15°C 30°C

D (cm 2 /s)

45°C

1.00E-7

1.00E-8 0.00

0.20

0.40

0.60

0.80

1.00

0.80

1.00

0.80

1.00

Ceq (wt%) 1.00E-6

PLA+1%ZnO 15°C 30°C

D (cm 2 /s)

45°C

1.00E-7

1.00E-8 0.00

0.20

0.40

0.60

Ceq (wt%) 1.00E-6

PLA 15°C 30°C

D (cm 2 /s)

45°C

1.00E-7

1.00E-8 0.00

0.20

0.40

0.60

Ceq (wt%)

Figure 6. Diffusion coefficient, D (cm2/s), as function of Ceq of water vapour (wt%) at different temperatures for neat PLA and nanocomposites loaded with 1% and 3% ZnO.

22

Permeability values to water vapor, reported in Table 5, were evaluated as product of sorption and diffusion following the Eq. 7: P=SxD

7

Noteworthy, as it comes out from the results shown in the Table 5, the increasing of temperature triggers an increasing of permeability, while at constant temperature this parameter does not show significant variation. Table 5. Permeability parameters to water vapour, P (wt%/atm x cm2/s), for PLA and nanocomposite films loaded with 1% and 3% ZnO Sample → PLA Temperature , °C↓

PLA-

PLA-

1% ZnO

3% ZnO

15

8.26 10-7

9.81 10-7

6.43 10-7

30

14.7 10-7

10.4 10-7

9.30 10-7

45

14.6 10-7

18.7 10-7

17.5 10-7

1.00E-6 PLA PLA+1% ZnO

2

Do (cm /s)

PLA+3% ZnO

1.00E-7

1.00E-8 3.00

3.10

3.20

3.30

1/T (K) x 10

3.40

3.50

3

Figure 7. Do (cm2/s) as function of 1/T (K).

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Antibacterial properties To assess the antimicrobial effects of ZnO, the nanocomposite films were evaluated against E. coli and S. aureus, respectively Gram-negative and Gram-positive bacteria. Samples of unfilled PLA and PLA-ZnO nanocomposites with a surface area of about 1 cm2 and thickness of ~ 150 µm were incubated with each bacterial suspension at 37 °C and constant agitation, harvested at different times and plated on a rich medium and incubated for 24 h. Subsequently, the colony forming units (CFU) were determined and the antibacterial activity (A) was measured as differences between logarithm values of growth of untreated (neat PLA) and treated (PLA-ZnO) samples. First of all, it is noteworthy mentioning that the mechanisms responsible for the antibacterial activity of ZnO nanoparticles are still not fully clear. However, the antibacterial effect is mainly ascribed to the photocatalytic generation of a large number of reactive oxygen species (superoxide anions and hydroxyl radicals, generation of hydrogen peroxide), to the formation of Zn2+ ions or the capture of ZnO onto the cell membrane, etc. [26, 50]. The values of antibacterial activity are summarized in Table 6. On one hand, for E. coli at 24 h it was found that in presence of ZnO the number of CFU decreased with respect to the control, but the antimicrobial effect was low (> 70 % of killed bacteria). On the other hand, after 7 days (see also Fig. 8a) the antibacterial effect was high for all concentrations and even very good for nanocomposite films containing low amount of nanofiller, i.e., 1% ZnO. In fact following the values of A that are higher than 2 after 7 days (Table 6) the antimicrobial performance is considered good or very good, which means a reduction of bacterial population of 99% or more. For sake of comparison, it is of interest to note that in the absence of ZnO, for the neat PLA the number of CFU after a similar incubation time is mostly unchanged (see Fig. 8a). 24

In S. aureus antibacterial properties occurred only with films containing 3% ZnO. Similarly to E. coli, significant reduction of population (>99%) was obtained after 7 days of treatment (A= 2.85) (Table 6 and Fig. 8b).

Table 6. Antibacterial activity (A) of selected PLA-ZnO films against E. coli and S. aureus after different incubation times Sample

Average (log CFU) 0h

24h

Growth24h

Growth 7 d (day)

7d

Antibacterial

Antibacterial

Activity (24 h) (A24h)

Activity (7 d) (A7d)

Escherichia coli PLA

6.76

6.59

6.62

F= -0.17

F = -0.14

(A24h=F24h–G%_24h)

(A7d = F7d – G%_7d)

PLA- 1% ZnO

6.66

5.89

3.20

G1.0% = -0.77

G1.0% = -3.46

0.60

3.32

PLA- 2% ZnO

6.76

5.84

4.38

G2.0% = -0.92

G2.0% = -2.38

0.75

2.24

PLA- 3% ZnO

6.70

6.00

4.30

G3.0% = -0.70

G3.0% = -2.40

0.53

2.26

Stapylococcus aureus PLA

7.16

7.08

4.98

F = -0.08

F=-2.18

(A24h = F24h – G%_24h)

PLA- 3% ZnO

7.11

6.81

2.08

G3.0% = -0.30

G3.0 = -5.03

0.22

(A7d = F7d – G%_7d) 2.85

Following these results, it is assumed that under the specific experimental conditions the antibacterial activities to both bacteria were time-dependent. Moreover, similarly to the behavior of other metal-polymer formulations [51], it is believed that the release of antimicrobial products, such as reactive oxygen species and/or of Zn2+ ions, can require longer time than 24h to obtain the minimum inhibitory concentration and optimal effectiveness. The different sensitivity of E. coli and S. aureus to the concentration of ZnO may be due to structural differences between the cell wall of Gram positive and Gram-negative bacteria. However this could also be due to the characteristic of S. aureus to form aggregates which would protect more internal cells from lower doses of antimicrobial products. Finally as remark, growth (G) values

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resulted significantly negative for all nanocomposites, as attended by a toxic effect of ZnO, however, slightly negative values were also found for control populations (F), indicating an absence of growth and a reduction of the microbial populations in experimental conditions. This occurred because to reduce interference of nanocomposite materials with the substances dissolved in growth media, microorganisms were suspended in distilled water (E. coli) or in diluted peptone water (S. aureus), both non suitable to support the bacterial growth. Nonetheless, under these conditions the bacteria tested may remain viable several weeks. Thus, based on antimicrobial activity, ZnO containing nanocomposites showed a high antibacterial effect toward analyzed Gram positive and negative microorganisms, but the bacterial sensitivity to them was different, with E. coli killed efficiently at concentrations apparently innocuous for Gram positive.

Mechanical properties and additional end-use characteristics Mechanical parameters were evaluated from stress-strain curves following the tensile tests performed at ambient temperature. As shown in Table 7, the addition of surface-treated ZnO into PLA leads to the slight increase of the rigidity (Young’s modulus, E) whereas both tensile stress at yield (σy) and at break (σb) reveal interesting values for packaging applications (e.g., σy is in the range 39 - 44 MPa) as compared to the neat PLA matrix (σy of 45 MPa). However, especially at high ZnO loading (3%), a slight reduction of the tensile stress was observed, that is reasonably associated to some diminution of molecular weights and to formation of low molecular products as aforementioned. Furthermore, like the neat PLA, the nanocomposite films show low elongation at yield (εy) and break (εb), without an apparent correlation with the nanofiller amount. To improve this parameter of interest in the perspective of further applications, the addition of a plasticizer into PLA-ZnO nanocomposite films can be supplementary considered.

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a

b Figure 8 (a, b). Evolution of log10 CFU (colony forming units) at various time intervals of E. coli (a) and S. aureus (b) on the surface of PLA and PLA-ZnO nanocomposite films

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Table 7: Comparative mechanical properties of PLA and PLA–ZnO nanocomposite films (standard deviations are given in brackets) Sample

E

σy

σb

εy *

εb*

(%- by weight)

(MPa)

(MPa)

(MPa)

(%)

(%)

PLA

2700 (±200)

45 (±3)

42 (±3)

2.5 (±0.3)

9.3 (±2.4)

PLA- 1% ZnO

2900 (±300)

44 (±5)

41 (±5)

3.3 (±0.7)

13.0 (±4.1)

PLA- 2% ZnO

3000 (±150)

42 (±4)

39 (±3)

1.9 (±0.1)

7.1 (±1.5)

PLA- 3% ZnO

2800 (±100)

39 (±2)

35 (±3)

2.0 (±0.3)

12.9 (±3.0)

*Distance between grips of 40 mm. The opacity is a parameter that must be considered for packaging applications, since it affects the appearance of the products. Figure 9 reports the experimental data obtained on films of about 150 micron thickness. The opacity is only slightly increased with a higher ZnO amount. The opacities were ~10% higher for the films containing 3% ZnO. Such effect, due to the interference of nanoparticles with the light, must be taken into account since this is relevant for packaging applications.

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Figure 9. Opacity of 150 micron-thick films vs. the ZnO content

Finally it is of interest to note that the additional investigations (thermogravimetric analyses (TGA) and UV–Visible absorption spectra, results not shown here) confirm the previously reported results [34], respectively, addition of ZnO is leading in some decrease of PLA thermal stability, whereas once more, at above 1% ZnO the films were characterized by a total anti-UV protection and good transmittance of the visible light (400–800 nm). By considering the multifunctional properties that are specific for PLA-ZnO films (mechanical performances, antiUV, antibacterial, barrier characteristics) with respect to PLA or other PLA nanocomposites and additional potential features, such as the possibility of utilization as self-cleaning materials, these new nanocomposites can present a high interest in traditional and special applications as biosourced packaging materials.

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4. Conclusions The work was focused on the production and characterization of PLA-ZnO films to highlight their key-characteristics. PLA-ZnO nanocomposites were produced by melt-compounding PLA and 0.5 - 3% surface-treated ZnO rod-like nanoparticles, step followed by the production of films. It was demonstrated that it is possible to obtain competitive thin films characterized by adequate ZnO dispersion and quite good preservation of molecular and mechanical properties particularly when the polyester/nanofiller interface is adequately tuned via a triethoxy caprylylsilane surface-treatment. According to DSC and consistent with XRD the produced films were mostly amorphous. They were analyzed with special attention paid to the water vapor barrier properties (sorption, diffusion, permeability) which resulted from the nanofiller incorporation. It was assumed that the well dispersed nanoparticles through the polyester matrix can provoke certain tortuosity to water vapor molecules. The changes in PLA inherent permeation properties were mainly dependent on the temperature and nanofiller loading. The values of energy activation were higher for the films containing 3% ZnO with respect to the neat PLA, confirming the increased difficulty for molecules to diffuse through the PLA matrix. The nanocomposite films were characterized by a good antibacterial activity against Gram-positive and Gram-negative bacteria and the antibacterial performance was found to be time-dependent. By considering the multifunctional properties of PLA-ZnO films (anti-UV, antibacterial, mechanical, etc.), such nanocomposites produced by extrusion could be considered as a very promising approach in the production of environmental-friendly packaging materials.

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Acknowledgements The authors from University of Salerno thank Ms Laura Bassi for carrying out part of the experiments of transport properties during her work of thesis for bachelor in Chemical Engineering. The authors from UMONS & Materia Nova thank the Wallonia Region, Nord-Pas de Calais Region and European Community for the financial support in the frame of the INTERREG – NANOLAC project. They thank to Yoann Paint and Anne-Laure Dechief for assistance in the preparation and characterization of samples, NANOLAC partners and all mentioned companies for supplying raw materials. CIRMAP acknowledges supports by the Région Wallonne in the frame of OPTI²MAT program of excellence, by the Interuniversity Attraction Pole program of the Belgian Federal Science Policy Office (PAI 6/27) and by FNRS-FRFC.

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Nanocomposites of PLA and surface-treated ZnO rod-like nanoparticles. Changes in PLA inherent permeation properties dependent on temperature and loading Good antibacterial activity against Gram-positive and Gram-negative bacteria The antibacterial performance was found to be time-dependent.

39