*2.2. Size Exclusion Chromatography*

Plastic NPs were analyzed by low-pressure size exclusion chromatography using the Bio-logic system (Biorad, Ontario, Canada). The column (40 cm × 1 cm) was filled with Sephacryl S-500 gel and equilibrated with 0.2% Tween-20 and 14 mM NaCl at pH 7.4 at a flow rate of 1 mL/min. Sephacryl S-500 is a high-resolution gel designed for the separation of large macromolecules between 2 × 107 to 4 × 104 g/mole size range and suitable to resolve plastic NPs from the protein/lipid pool. Conductivity (NaCl and other salts) and absorbance at 280 nm (proteins) were continuously monitored. One mL fractions (0.75 mL/min) were collected for NR staining (10 μM final concentration). Clear and fluorescently labeled (yellow–green Fluoresbrite) polystyrene nanoparticles of 50 and 100 nm were purchased at Polyscience Inc. (Warrington, PA, USA) and were used for column calibration along with NaCl and albumin (66 kDa).

Calibration was achieved using fluorescently labeled polystyrene NPs in the absence/presence of biological sample (proteins) to determine the optimal separation conditions. As plastic NPs tend to adsorb proteins and lipids, forming a corona at the surface, various loading and elution buffers were tested to find optimal conditions at the levels of detergent type (ionic vs. nonionic detergents), concentration, solvent (acetonitrile, acetone, and ethanol) and salt concentration (NaCl). It was found that the addition of albumin and tissue homogenate extracts (proteins/lipids) interacted with nanoparticles in low salt

(<10 mM NaCl) and detergent concentrations (<0.05%). Incubating the homogenates fractions in high salts (0.7 M NaCl) in the presence of nonionic detergent (0.05–0.2% Tween-20) in the sample buffer and nonionic detergent (0.2% Tween-20 in 14 mM NaCl) for the elution buffer eliminated nanoparticle–protein interactions and facilitated separation of plastic NPs from the proteins/lipids pool.

Homogenate fractions were either directly injected in the column or previously fractionated by a salting-out methodology using saturated NaCl/ACN. For the direct injection, the homogenate supernatants (3000× *g* 10 min) were thawed on ice and mixed with one volume of 1.4 M NaCl and 0.2% Tween-20 buffered at pH 7.4 (with1MK2HPO4) for 5 min. As explained above, the addition of salts and nonionic detergents were necessary to keep proteins from adhering to the nanoparticle's surface and to exclude suspended plastic particles (density). For the salting-out step, 300 μL of the homogenate was mixed with 400 μL of 5 M NaCl and 400 μL of ACN. The sample was mixed and centrifuged at 3000× *g* for 10 min as described above. The ACN upper phase was mixed with 0.1 volume of 1.4 M NaCl/Tween-20 0.1% and injected to the chromatography column (0.25 mL) using the same elution buffer (0.2% Tween-20, 14 mM NaCl). A volume of 0.25 mL of transparent or fluorescent plastic nanoparticles (50 and 100 nm), albumin (66 kDa), and 0.5 M NaCl (total volume) was also injected in the column for calibration. The flow rate was 0.75 mL/min and 1 mL fractions were collected to a total volume of 30 mL. The absorbance at 280 nm (proteins and polystyrene) and conductivity (mS) were continuously measured.

The collected fractions were stained with 10 μM NR dye (from a 100 μM stock solution prepared in 0.2% Tween-20 and 10% ethanol in 14 mM NaCl) and fluorescence was measured at 485 nm excitation and 530 nm (40 nm bandpass) (Turner Biosystems, Sunnyvale, CA, USA). The blank consisted of the elution buffer, and increasing concentrations (0.05–1 μg/mL) of 50 nm diameter transparent polystyrene NPs (Polyscience Inc., Warrington, PA, USA) were used for calibration. The elution volume of the analyte (Ve) was determined based on NR fluorescence, and total volume (Vt) of the column was determined by NaCl conductivity spike from the sample buffer. The void volume (Vo) was based on the supplier information (around 24% of the total column volume = 7.5 mL). The elution profiles data were expressed as Ve/Vt, which permits comparisons with other column volumes.
