Images obtained after the characterisation of polylactic acid (PLA) nanoplastics by TEM and SEM

PLA-MNPLs obtention MNPL production requires a methodology allowing to get particles with a narrow size distribution, control of the morphology, and to be reproducible and robust. To obtain MNPLs with chemical characteristics like the particles that can be found in the environment, the top-bottom strategy was chosen starting from commercial-grade polymers in the form of pellets. In this way, we started with an additive polymer ready to obtain final plastic products. The following products were used, Pluronic® F-127 BioReagent and polyvinyl alcohol (PVA) (from Sigma-Aldrich). Dichloromethane (DCM), dried (max. 0.005 % H2O) ExpertQ® provided by Scharlab. Commercial grade of polylactic acid in pellet form. The 5(6)-FAM (5-(and-6)-carboxyfluorescein), SE (succinimidyl ester), and mixed isomers (fluorophore compound) were purchased from ThermoFisher Scientific. Solvent evaporation, combined with the miniemulsion technique was the methodology used to obtain the PLA reference material. The procedure was like the one reported by other groups (Feng et al., 2018; Chen et al., 2020). A pre-mini emulsion was prepared by adding the aqueous phase consisting of dissolved pluronic and PVA (as co-stabilizer) in 120 g water (0.25 % wt and 2 % wt, respectively) to the organic phase composed of 1 g of PLA dissolved in 30 g DCM and stirring magnetically for 60 min. Furthermore, ultrasonication under ice cooling was applied for 120 s at 80 % amplitude, using a Bandelin Electronic UW2200 sonicator. The obtained miniemulsion was transferred to a round bottom flask to evaporate the organic solvent, under pressure. The labeled PLA-NPLs were obtained in the same way as the unlabeled PLA-NPLs explained above. Previously, the functionalization of PLA was necessary using a reactive extrusion process without the use of solvents. Once PLA was functionalized with amino groups, it was labeled by reacting with the fluorophore compound. For this purpose, 1 g of amino-functionalized PLA was placed in a round-bottom flask provided with a magnetic stirring bar, an N2 inlet, and a reflux condenser. 12 mL of tetrahydrofuran (THF) was added under an inert atmosphere, and when the material was completely dissolved, 25 mg (2.5 % w/w) of 5(6)-FAM, SE was added. The reaction mixture was stirred at room temperature and protected from light. After 1 h, water was added to precipitate the labeled PLA, which was filtered off, washed with water, and dried. At this point, the fluorophore compound is chemically anchored to the functionalized PLA, and ready to be used.

PLA-NPLs physicochemical characterization Before use in Drosophila, PLA-NPLs were widely characterized using a set of approaches. Shape, morphology, and degree of aggregation of PLA-NPLs were determined using both scanning electron microscopy (SEM) (Zeiss Merlin, Zeiss, Oberkochen, Germany) and a TEM instrument (JEOL JEM 1400, JEOL LTD, Tokyo, Japan). The transparency nature of PLA obstructed TEM investigation since the particles showed a low contrast. This was overcome by staining particles with lead acetate. To stain PLA-NPLs, 20 μL of a PLA-NPLs dispersion (2 mg/mL) was loaded upon a copper grid covered with carbon film, and the dried samples were stained with 20 μL of lead acetate. For SEM investigation, the PLA-NPLs suspension was loaded on the bright surface of cleaned silicon chips. Silicon chips were cleaned with drops of Milli-Q water and kept in a clean area. The average diameter of PLA-NPLs was determined by measuring 100 random particles of SEM images using the ImageJ software. The functional groups of PLA particles were detected with Fourier transform infrared spectroscopy (FTIR). A Zetasizer® Ultra device from Malvern Analytical (Cambridge, United Kingdom) was used to evaluate the hydrodynamic size (dynamic light scattering, DLS) as well as the total surface charge of PLA-NPLs (Zeta potential) in suspension. FTIR, DLS, and zeta potential were measured at the Molecular Spectroscopy and Optical Microscopy unit of the Institut Català de Nanociència i Nanotecnologia (ICN2) at the UAB campus.