Incineration of Nanoclay Composites Leads to Byproducts with Reduced Cellular Reactivity

Addition of nanoclays into a polymer matrix leads to nanocomposites with enhanced properties to be used in plastics for food packaging applications. Because of the plastics’ high stored energy value, such nanocomposites make good candidates for disposal via municipal solid waste plants. However, upon disposal, increased concerns related to nanocomposites’ byproducts potential toxicity arise, especially considering that such byproducts could escape disposal filters to cause inhalation hazards. Herein, we investigated the effects that byproducts of a polymer polylactic acid-based nanocomposite containing a functionalized montmorillonite nanoclay (Cloisite 30B) could pose to human lung epithelial cells, used as a model for inhalation exposure. Analysis showed that the byproducts induced toxic responses, including reductions in cellular viability, changes in cellular morphology, and cytoskeletal alterations, however only at high doses of exposure. The degree of dispersion of nanoclays in the polymer matrix appeared to influence the material characteristics, degradation, and ultimately toxicity. With toxicity of the byproduct occurring at high doses, safety protocols should be considered, along with deleterious effects investigations to thus help aid in safer, yet still effective products and disposal strategies.


Nanocomposite Preparation
Polylactic acid 6752 (PLA) was melt-mixed with Cloisite 30B (CC) loaded at a 5 wt. %, in a Thermo-Haake internal mixer operating at 200 ºC and 80 rpm for 5 min. Thin films were then molded at 200 ºC using a compression press to form PLA-CC nanocomposites (PLACC), as well as PLA films to be used as controls.

Thermal Degradation of PLACC and CC
PLACC (1 g per sample) and CC (0.5 g per sample) were thermally degraded using a TGA701 Thermogravimetric Analyzer (LECO). To determine the moisture content, the samples were heated in nitrogen at a rate of 6 ºC/min and in a range of temperatures from 25 ºC to 105 ºC. To determine the volatile content, the samples were heated from 105 ºC to 950 ºC also in nitrogen and at a rate of 43 ºC/min. Finally, to determine the ash content, the samples were heated from 550 ºC to 900 ºC in oxygen, at a rate of 15 ºC/min.

Material Characterization of PLA, PLACC, and Associated Nanoclays or Byproducts
Molecular composition of CC, thermally degraded CC (CC900), and thermally degraded PLACC (PLACC900) was determined using Fourier Transform Infrared Spectroscopy (FTIR, Digilab FTS 7000) equipped with diamond Attenuated Total Reflection (ATR). Scans were collected in the range of 4000-400 cm -1 at a resolution of 4 cm -1 ; a total of 100 scans were coadded to form the final spectrum for each of the samples. Elemental composition of PLACC900 and CC900 was investigated using a Hitachi S-4700 Field Emission Scanning Electron Microscope (SEM, Hitachi High-Technologies Corporation) equipped with energy dispersive X-ray (EDX) spectroscopy at 20.0 kV.
The absorption spectra for PLA and PLACC was determined in the range of 200-800 nm via the Shimadzu UV-Vis spectrophotometer (Shimadzu Scientific Instruments). UV barrier properties of the film were determined by measuring transmission at 280 nm, and transparency of the films was determined by measuring transmission at 660 nm, also via the Shimadzu UV-Vis spectrophotometer. The tensile strength, Young's Modulus, and elongation at break for films of PLA and PLACC were evaluated via the Instron E1000 (Instron Corporation) under a 2 kN load cell and using the Bluehill 3 software. For this, rectangular films of PLA and PLACC, 5 mm in width x 32 mm in length x 0.3 mm in thickness, were placed in the Instron grips, and the experiments were performed with a crosshead speed set at 5 mm/min. A specimen gauge length of about 25 mm was used for each sample upon gripping in the crosshead.
The size distribution of PLACC900 was determined by dynamic light scattering (DLS) via the Mastersizer 2000 with a Hydro 2000S accessory (Malvern Instruments). For this, solutions of PLACC900 dispersed and bath sonicated in cell culture media (Dulbecco's Modified Eagle Medium: DMEM) containing 5% fetal bovine serum (FBS) or in phosphate buffered saline (PBS), were dropped into the Hydro 2000S until laser obscuration was within 10-20 %. The size analysis was performed 3 consecutive times with a stirrer speed of 1750 rpm and under continuous sonication. To note: DLS predicts size measurements of particles by applying models to the light that is being captured from the scattering of the particles. Specifically, the Mie theory is used to interpret the scattering patterns of light by the particles and assumes that particles are perfect spheres. In order to account for any changes in shape, the Mastersizer 2000 uses the technique known as "equivalent spheres" and instead of measuring the size of the particles it measures the volume they occupy. 3

Live Cell Count
BEAS-2B cells were seeded in a 12 well plate (Falcon) at a density of 2.0x10 5 cells/ml. After 24 h, the cells were exposed to PLACC900 at 100 and 300 µg/ml, which were first sonicated for 8-10 min in media via a bath sonicator (Branson). Cells in only media served as controls. Twenty four, 48, and 72 h post exposure to PLACC900 the cells were trypsinized and stained with 0.4% trypan blue solution. Subsequently, 10 µl of the sample containing the stained cells was added to a hemocytometer, and the number of cells in the 4 outer grids were counted through use of the Leica DM IL optical microscope using a 10X objective.

Reactive Oxygen Species (ROS) Generation of PLACC900
PLACC900 was dispersed in media via a bath sonicator at doses of 100 and 300 µg/ml. The solutions were then placed in a 12 well plate with media only serving as the control. After 24, 48, and 72 h of incubation at 37 ºC, 5 % CO2, and 80 % relative humidity, 50 µl of the PLACC900+media from each dose (and media only as control) was transferred to a blackbottomed 96 well plate. Subsequently, 50 µl of PBS and 50 µl of Lumigen ECL Plus (Lumigen, Inc.) were added to each well, and the samples were incubated for 5 min in the dark. Luminescence was read at 600 nm via the FLUOstar OPTIMA plate reader.