Surface Chemistry-Dependent Evolution of the Nanomaterial Corona on TiO2 Nanomaterials Following Uptake and Sub-Cellular Localization

Nanomaterial (NM) surface chemistry has an established and significant effect on interactions at the nano-bio interface, with important toxicological consequences for manufactured NMs, as well as potent effects on the pharmacokinetics and efficacy of nano-therapies. In this work, the effects of different surface modifications (PVP, Dispex AA4040, and Pluronic F127) on the uptake, cellular distribution, and degradation of titanium dioxide NMs (TiO2 NMs, ~10 nm core size) are assessed and correlated with the localization of fluorescently-labeled serum proteins forming their coronas. Imaging approaches with an increasing spatial resolution, including automated high throughput live cell imaging, correlative confocal fluorescence and reflectance microscopy, and dSTORM super-resolution microscopy, are used to explore the cellular fate of these NMs and their associated serum proteins. Uncoated TiO2 NMs demonstrate a rapid loss of corona proteins, while surface coating results in the retention of the corona signal after internalization for at least 24 h (varying with coating composition). Imaging with two-color super-resolution dSTORM revealed that the apparent TiO2 NM single agglomerates observed in diffraction-limited confocal microscopy are actually adjacent smaller agglomerates, and provides novel insights into the spatial arrangement of the initial and exchanged coronas adsorbed at the NM surfaces.


Equipment
-Microbalance with accuracy of 0.1 mg or better -Laminar flow Biosafety cabinet to ensure sterile handling of the materials -Sterile 15 ml conical polystyrene tubes -Micropipettes + sterile tips -Vortex mixer -Ultrasonic bath (min. 250 Watt), JRC will use a vial tweeter -For endotoxin test: sterile microvials and refrigerated microcentrifuge 2. Dispersion and dilution of powdered MNMs -The stock dispersions and dilutions should be prepared freshly (30 -60 min before adding to the cells). This is especially important for partly soluble MNMs like ZnO or Ag.
-weigh the powder and prepare a dispersion of 5 mg/ml in sterile water, -vortex and ultrasonicate the dispersion for 15 min in an ultrasonic bath -Prepare pre-warmed medium containing 10% of the NanoMile centralized serum and dilute the stock suspension (serial dilution) to the concentrations to be tested in this medium (125, 62.5, 31.3, 15.6, 7.8, 3.9, 2.0, 1.0 µg/ml). The suspension has to be vortexed vigorously immediately before taking out an aliquot.
-Mix the diluted dispersions again immediately before adding to the cells, either by vortexing or pipetting up and down.

Modification for hydrophobic material according the NanoGenotox protocol:
-After weighing the powder pre-wet the material with ethanol (96% or higher). Calculate the ethanol volume for a 0.5% (v/v) ethanol concentration in the 5 mg/mL stock suspension.
-Proceed with the suspension procedure as described above.

Dilution of suspended MNMs (stocks > 5 mg/ml in water)
-Freshly prepare a working dispersion of 5 mg/mL in sterile water from the dispersion delivered, which has to be vortexed vigorously immediately before taking out an aliquot. -Particle dispersions are vortexed and ultrasonicated for 5 min in an ultrasonic bath.
-Prepare pre-warmed medium containing 10% of the NanoMILE centralized serum and dilute the stock suspension (serial dilution) to the concentrations to be tested in this medium (125, 62.5, 31.3, 15.6, 7.8, 3.9, 2.0, 1.0 µg/ml). The suspensions have to be vortexed vigorously immediately before taking out an aliquot. -Mix the diluted dispersions again immediately before adding to the cells, either by vortexing or pipetting up and down.
-If the stocks are lower concentrated than 5 mg/ml dilution can be done in medium plus 10% serum directly.

Endotoxin test (protocol used at KIT)
-An aliquot of the particle stock suspension (5 mg/ml in water) is centrifuged at 20,000x g for 10 min. Particles of low density e.g. polystyrene must be centrifuged for 60 min minimum. -The supernatant is used for the endotoxin test according the instructions from the test kit supplier.   Figure 5 (A) FITC labelling of serum containing media results in the effective labelling of the protein corona in cells treated with uncoated and dispex-44040 coated TiO 2 NMs. FITC-serum signal is significantly higher in cells treated with NMs compared to untreated controls, however some discernable non-NM associated FITC signal is detectable. (B) As such quantification was performed by specifically measuring FITC associated with reflectance signal, thereby robustly limiting FITC-serum measurements to the NM corona. FITCserum signal measured was significantly different compared to controls according to a One Way ANOVA (p < 0.0001) n = 3. (C) Co-localization between FITC and Reflectance Signal was shown to be significantly higher when compared to controls (* p = 0.0202). For an effective automated analysis, cell trace and FITC signal were first de-noised and segmented to generate binaries (cell trace binaries in blue, FITC binaries in red with green outlines). Cell trace binaries were also subject to post-processing, where cells touching image borders were removed, as well as cells which could not be effectively watershed. Following this initial segmentation step, a second set of binaries were generated which were comprised of cell trace binaries positive for FITC binaries, indicated in yellow. The sum FITC intensity in these fields was calculated and subsequently divided by the total cell trace area (blue binaries). This allows for an effective, automated segmentation of the cell volume, as well as the detection of cell associated FITC signal. Secondly, combined binaries are used to restrict analysis to masks within specific regions of interest. For instance, once reflectance masks have been generated for the whole field, a second binary is produced based on reflectance masks positive for Cell Trace signal, thereby allowing for a robust analysis of NMs specifically within the cell volume. (C) This approach can be then applied to study FITC signal specifically associated with reflectance binaries, and thereby effectively remove background signal which is not associated with NMs. By measuring these restricted binaries and normalising the subsequent intensity data to the Cell Trace measured area of the cell, comparable FITC NM corona intensities can be measured. This approach is also applied to determine the co-localisation of NMs with lysosomes, where binaries positive for both lysosomal and reflectance signal are calculated as a percentage of the total number of NMs internalised. Similarly, mean reflectance intensity is most significantly increased for TiO 2 -F127 and TiO 2 -AA4040 at 18 hours, and decreases slightly at 24, suggesting more tightly bound agglomerates at 18 hours. (E) Finally, no significant difference is observed in lysosomal co-localisation across TiO 2 NMs, however a significant increase is observed over time which is consistent with trafficking to these compartments.