Difference in Endocytosis Pathways Used by Differentiated Versus Nondifferentiated Epithelial Caco-2 Cells to Internalize Nanosized Particles

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells’ internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.


Figure S6. The effect of endocytosis inhibitors in nanoparticles internalization experiment on the transepithelial electrical resistance (TEER) of Caco-2 cell layers.
Cell layers were pre-incubated for 30 minutes with the inhibitors at the apical side, followed by 180 minutes of exposure to the nanoparticles (50 µg/ml), in the presence of the same inhibitors.TEER was measured every 30 minutes over 180 minutes, followed by sample removal and addition of the culture medium.After incubation for 1440 minutes (24 hours) in normal cell culture conditions (37°C), TEER was measured again to determine the recovery.Results are displayed as a % of the control (inhibitor-free) TEER value.Data represent the mean ± SD (n = 4).

Figure S7. The effect of pharmacological inhibitors on cholera-β-toxin (CβT) transferrin (TF) uptake in non-polarized cells. a) -d) nystatin, filipin, MβCD and genistein effect on internalisation of cholera-β-toxin in
non-polarised Caco-2 cells.Cells were pre-treated with the inhibitors for 30 min, followed by 180 min of exposure to Alexa fluor labelled cholera-β-toxin (CβT) in the presence of the respective inhibitor; e) effect of chlorpromazine on internalisation of transferrin (TF), cells were pre-treated with the inhibitor for 30 min, followed by 180 min of exposure to Alexa fluor labelled transferrin in the presence of the inhibitor.Uptake is expressed as % of the control (inhibitor-free) value.*** indicates p<0.001 a statistically difference compared to control (untreated cell layers).

Comments:
In this study a treatment with filipin at non-toxic concentration of 2 μg/ml was not efficient in inhibiting caveolae-mediated endocytosis of cholera-β-toxin.It has been reported that treatment of Caco-2 cells with 5 μg/ml filipin can reduce the uptake of cholera-βtoxin by about 20% (DOI: 10.1242/jcs.114.20.3737), but in our study this concentration showed a significant cell toxicity.Despite this observation, we proceeded with filipin treatment in the view of reported complexity and different observations of cholera-β-toxin pathways of cellular internalisation in different cells (DOI: 10.1242/jcs.114.20.3737), in addition to using another cholesterol affecting compound, nystatin, in our study.

Figure S1 .
Figure S1.TEER profile of Caco-2 cells cultured on permeable inserts.Background TEER due to the permeable membrane inserts was subtracted from the reported TEER values.Data are presented as the mean ± SD (n = 10 to12).

Figure S2 . 7 Figure S3 .
Figure S2.Scanning electron microscopy images of polystyrene nanoparticles used in the study.

Figure S5 .Filipin
Figure S5.Relative cell viability (%) after incubation of Caco-2 cells with different endocytosis inhibitors for 4 hours.Data are the results of an MTS assay; they are expressed as relative cell metabolic activity and presented as the mean ± SD (n =7).

Figure S8 :
Figure S8: The effect of pharmacological inhibitors on internalisation of transferrin (TF) and choleraβ-toxin (CβT) in polarized Caco-2 cell layers.The cell layers were pre-treated with an inhibitor solution for 30 min, followed by 120 min and 180 min of exposure to Alexa fluor labelled transferrin or Alexa fluor labelled cholera-β-toxin (CβT) in the presence of the inhibitor.* and ** Indicate p<0.05 and p<0.01, respectively, a statistically difference compared to control (untreated cell layers) whereas ns indicates non-significant.

Table S2 .
Selected concentrations of inhibitors and their recognised effect on cell internalization pathways