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. 2011 Apr 26;5(4):2756-2769.
doi: 10.1021/nn200328m. Epub 2011 Mar 2.

Differential expression of syndecan-1 mediates cationic nanoparticle toxicity in undifferentiated versus differentiated normal human bronchial epithelial cells

Haiyuan Zhang #  1 , Tian Xia #  2 , Huan Meng  2 , Min Xue  3 , Saji George  1   2 , Zhaoxia Ji  1 , Xiang Wang  1 , Rong Liu  4 , Meiying Wang  2 , Bryan France  5 , Robert Rallo  4 , Robert Damoiseaux  1   6 , Yoram Cohen  4 , Kenneth A Bradley  1   5 , Jeffrey I Zink  3 , Andre E Nel  1   2
Affiliations

Differential expression of syndecan-1 mediates cationic nanoparticle toxicity in undifferentiated versus differentiated normal human bronchial epithelial cells

Haiyuan Zhang et al. ACS Nano. .

Abstract

Most in vitro toxicity studies on engineered nanomaterials (ENMs) use transformed rather than primary cells for logistical reasons. However, primary cells may provide a more appropriate connection to in vivo toxicity because these cells maintain their phenotypic fidelity and are also capable of differentiating into lineages that may be differently affected by potentially hazardous ENMs. Few studies to date have focused on the role of cellular differentiation in determining ENM toxicity. We compared the response of undifferentiated and differentiated primary human bronchial epithelial (NHBE) cells to cationic mesoporous silica nanoparticles (MSNPs) that are coated with polyethyleneimine (PEI) since this polymer is known to exert differential cytotoxicity depending on its molecular weight and cationic density. The attachment of cationic PEI polymers to the MSNP surface was used to assess these materials' toxicological potential in undifferentiated and differentiated human bronchial epithelial cells, using a multiparametric assay that screens for an integrated set of sublethal and lethal response outcomes. MSNPs coated with high molecular weight (10 and 25 kD) polymers were more toxic in differentiated cells than particles coated with shorter length polymers. The increased susceptibility of the differentiated cells is in agreement with more abundant expression of a proteoglycan, syndecan-1, which contains copious heparin sulfate side chains. Pretreatment with heparinase to remove the negatively charged sulfates decreased MSNP-PEI binding to the cell surface and lowered the cytotoxic potential of the cationic particles. These data demonstrate the importance of studying cellular differentiation as an important variable in the response of primary cells to toxic ENM properties.

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Figures

Figure 1
Figure 1. TEM images of PEI-MSNPs
TEM images were obtained prior to and after coating with the 25, 10 and 1.8 kD PEI polymer. The bottom panels show the pores on the coated and uncoated particles remain open. Other PEI-coated particles had the same appearance. Scale bar, 100 nm.
Figure 2
Figure 2. Confocal microscopy showing the undifferentiated and differentiated cell morphologies in parallel with immunoblotting results for epithelial differentiation markers
(A) Confocal microscopy showing difference in cellular morphology in undifferentiated (left) and differentiated cells (right). The cell membrane was stained by Alexa Fluor 594-conjugated wheat germ agglutinin (WGA) and nuclei stained by Hoechst 33342. Cells were visualized under a Leica confocal 1P/FCS microscope. (B) Immunoblotting analysis comparing SPR-2 and β-catenin expression in undifferentiated and differentiated cells relative to the household pattern, β-actin.
Figure 3
Figure 3. Heatmap to compare the toxicity of various PEI-coated particles in undifferentiated and differentiated NHBE by a rapid throughput multi-parametric assay
The heatmap was established using SSMD statistical analysis to evaluate significant differences in the toxicological response profiles of undifferentiated and differentiated NHBE. The response parameters included in the multi-parametric assay includes measurement of intracellular calcium flux (Fluo-4), superoxide generation (MitoSox Red), H2O2 generation (DCF) and mitochondrial membrane depolarization (JC-1). Cells were treated by MSNP-PEI or MSNP at doses of 0.4, 0.8, 1.6, 3.2, 6.3, 12.5, 25, 50, 100, 200 μg/mL. Epifluorescence images were collected hourly for 6 hr. The layout of the 384 well plate used in this experimentation is shown in Fig. S7. Full details of the multi-parametric assay, preparation of the particles and processing of the plate appear in materials and methods. The fluorescent dyes are displayed in Table 3.
Figure 4
Figure 4. Differential cellular association of FITC-labeled MSNP-PEI in undifferentiated and differentiated NHBE
(A) FITC-labeled, PEI-coated MSNPs (25 μg/mL) were used to determine cellular association at 3 h by flow cytometry and confocal microscopy. The flow measurement used mean fluorescence intensity (MFI) units to study the cellular association of the particles. The MFIs of the particles coated with the 10 and 25 kD polymers is significantly higher in differentiated cells compared to undifferentiated cells; (B) Confocal microscopy images showing the cellular localization of FITC-MSNP-PEI 10 kD in undifferentiated and differentiated cells. A significant fraction of the labeled particles are attached to the surface membrane of differentiated cells. *p < 0.05 compared with control.
Figure 4
Figure 4. Differential cellular association of FITC-labeled MSNP-PEI in undifferentiated and differentiated NHBE
(A) FITC-labeled, PEI-coated MSNPs (25 μg/mL) were used to determine cellular association at 3 h by flow cytometry and confocal microscopy. The flow measurement used mean fluorescence intensity (MFI) units to study the cellular association of the particles. The MFIs of the particles coated with the 10 and 25 kD polymers is significantly higher in differentiated cells compared to undifferentiated cells; (B) Confocal microscopy images showing the cellular localization of FITC-MSNP-PEI 10 kD in undifferentiated and differentiated cells. A significant fraction of the labeled particles are attached to the surface membrane of differentiated cells. *p < 0.05 compared with control.
Figure 5
Figure 5. Surface membrane depolarization and the RBC hemolysis by cationic MSNP
(A) Assessment of surface membrane potential (SMP) using a fluorescence assay in which differentiated cells incubated with MSNP-PEI 25 kD and 10 kD and labeled with the FMP dye show a significant loss of membrane potential. The same particles had minimal effects in undifferentiated cells. Particles coated with the 1.8 kD polymer or non-coated particles had little effect. These data are in agreement with the cytotoxicity analysis in Fig. 3. (B) Hemolysis assay using mouse RBCs: while MSNP-PEI 10kD showed dose-dependent hemolysis (up to 14.9% of RBC) over the concentration range of 25–200 μg/mL, non-coated particles were without an effect. *p < 0.05 compared with control.
Figure 5
Figure 5. Surface membrane depolarization and the RBC hemolysis by cationic MSNP
(A) Assessment of surface membrane potential (SMP) using a fluorescence assay in which differentiated cells incubated with MSNP-PEI 25 kD and 10 kD and labeled with the FMP dye show a significant loss of membrane potential. The same particles had minimal effects in undifferentiated cells. Particles coated with the 1.8 kD polymer or non-coated particles had little effect. These data are in agreement with the cytotoxicity analysis in Fig. 3. (B) Hemolysis assay using mouse RBCs: while MSNP-PEI 10kD showed dose-dependent hemolysis (up to 14.9% of RBC) over the concentration range of 25–200 μg/mL, non-coated particles were without an effect. *p < 0.05 compared with control.
Figure 6
Figure 6. Cellular uptake and cytotoxicity in response to cationic MSNP after heparinase treatment
Undifferentiated and differentiated cells were treated with 5 U of heparinase I/II for 2 h, followed by exposure to FITC-labeled MSNP-PEI 10 kD and 25 kD (25 μg/mL) for 3 h. (A) MFI as determined by flow cytometry shows a 6.4-fold (MSNP-PEI 10 kD) or 6.8-fold (MSNP-PEI 25 kD) decrease in cellular association in heparinase-treated differentiated cells. However, there was no obvious difference in undifferentiated cells; (B) Flow cytometry to assess PI uptake shows a 3.4-fold (MSNP-PEI 10 kD) or 3.7-fold (MSNP-PEI 25 kD) decrease in the % PI stained cells in the differentiated population but no obvious decrease in undifferentiated cells following heparinase treatment; (C) Confocal microscopy showing decreased cell surface binding of FITC-labeled MSNP-PEI 10 kD in differentiated NHBE after heparinase treatment. However, there was no obvious difference in undifferentiated NHBE. *p < 0.05 compared with control.
Figure 6
Figure 6. Cellular uptake and cytotoxicity in response to cationic MSNP after heparinase treatment
Undifferentiated and differentiated cells were treated with 5 U of heparinase I/II for 2 h, followed by exposure to FITC-labeled MSNP-PEI 10 kD and 25 kD (25 μg/mL) for 3 h. (A) MFI as determined by flow cytometry shows a 6.4-fold (MSNP-PEI 10 kD) or 6.8-fold (MSNP-PEI 25 kD) decrease in cellular association in heparinase-treated differentiated cells. However, there was no obvious difference in undifferentiated cells; (B) Flow cytometry to assess PI uptake shows a 3.4-fold (MSNP-PEI 10 kD) or 3.7-fold (MSNP-PEI 25 kD) decrease in the % PI stained cells in the differentiated population but no obvious decrease in undifferentiated cells following heparinase treatment; (C) Confocal microscopy showing decreased cell surface binding of FITC-labeled MSNP-PEI 10 kD in differentiated NHBE after heparinase treatment. However, there was no obvious difference in undifferentiated NHBE. *p < 0.05 compared with control.
Figure 6
Figure 6. Cellular uptake and cytotoxicity in response to cationic MSNP after heparinase treatment
Undifferentiated and differentiated cells were treated with 5 U of heparinase I/II for 2 h, followed by exposure to FITC-labeled MSNP-PEI 10 kD and 25 kD (25 μg/mL) for 3 h. (A) MFI as determined by flow cytometry shows a 6.4-fold (MSNP-PEI 10 kD) or 6.8-fold (MSNP-PEI 25 kD) decrease in cellular association in heparinase-treated differentiated cells. However, there was no obvious difference in undifferentiated cells; (B) Flow cytometry to assess PI uptake shows a 3.4-fold (MSNP-PEI 10 kD) or 3.7-fold (MSNP-PEI 25 kD) decrease in the % PI stained cells in the differentiated population but no obvious decrease in undifferentiated cells following heparinase treatment; (C) Confocal microscopy showing decreased cell surface binding of FITC-labeled MSNP-PEI 10 kD in differentiated NHBE after heparinase treatment. However, there was no obvious difference in undifferentiated NHBE. *p < 0.05 compared with control.
Figure 7
Figure 7. Confocal microscopy and immunoblotting looking at syndecan-1 expression in undifferentiated and differentiated cells
(A) The cell membrane was stained by Alexa Fluor 594-conjugated wheat germ agglutinin (WGA) while nuclei were stained with Hoechst 33342. Sydecan-1 was detected by mouse anti-human syndecan-1 monoclonal antibody, which was secondarily detected by a green-fluorescent FITC-labeled goat anti-mouse antiserum. The combined panels on the right hand side demonstrate that syndecan-1 is abundantly expressed on the surface membrane. (B) Immunoblotting to show the enhanced expression of syndecan-1in differentiated NHBE compared to undifferentiated NHBE.
Figure 7
Figure 7. Confocal microscopy and immunoblotting looking at syndecan-1 expression in undifferentiated and differentiated cells
(A) The cell membrane was stained by Alexa Fluor 594-conjugated wheat germ agglutinin (WGA) while nuclei were stained with Hoechst 33342. Sydecan-1 was detected by mouse anti-human syndecan-1 monoclonal antibody, which was secondarily detected by a green-fluorescent FITC-labeled goat anti-mouse antiserum. The combined panels on the right hand side demonstrate that syndecan-1 is abundantly expressed on the surface membrane. (B) Immunoblotting to show the enhanced expression of syndecan-1in differentiated NHBE compared to undifferentiated NHBE.
Figure 8
Figure 8. Confocal microscopy to show the co-localization of syndecan-1 with FITC-labeled MSNP-PEI in differentiated cells
Syndecan-1 was detected by an antibody coupled to Alexa Fluor 594 (red fluorescence). FITC-labeled MSNP-PEI 10 kD (25 μg/mL) was added to the cells for 3 hr prior to protein and nuclear staining as in Fig. 7A. The combined panel on the right hand side demonstrates particle co-localizing with syndecan-1. The extent of the co-localization was 64.3% as determined by Image J software.
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