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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Nat Nanotechnol. 2020 Jun 1;15(7):605–614. doi: 10.1038/s41565-020-0693-6

Figure 8. DSPE-PCB micelles show no significant cytotoxicity or leaky gut-related endotoxin leakage and inflammation of repeated consecutive dosing.

Figure 8.

(a) Caco-2 cells were incubated with DSPE-PCB and Polysorbate 80 micelles with concentration range of 0.01–1 mg/mL for 24 h. Cell viability was measured in an MTT assay (Mean ± SD, N=6 biologically independent samples). (b) The treated Caco-2 cells were further visualized using live/dead staining (Calcein AM (green) and EthD-1 (red), respectively). Scale bar: 400 μm. Experiments were repeated three times independently with similar results. (c) Cell membrane integrity test by measuring lactate dehydrogenase (LDH) leakage. Caco-2 cells were co-cultured with DSEP-PCB, Polysorbate 80 for 24 hours, and the LDH levels were measured using LDH assay kit (Sigma) and plotted as % of untreated cell negative control (N=6 biologically independent samples, mean ± SD). As a positive control, 1% Triton X-100 and sonication were used to completely lyse the caco-2 cells and its LDH leakage level was found to be 240%. (d) Healthy mice were administered with DSPE-PCB micelle or saline through oral gavage twice daily for 14 consecutive days, followed by the intestinal permeability test. One hour after co-administering lactulose, mannitol, and DSPE-PCB micelles through ileum injection, urine was collected to measure the ratio of lactulose and mannitol contents (N=4 biologically independent samples, mean ± SD). A two-tailed t-test analysis was used for statistical analysis (NS: not significant). (e) Small intestine tissue sections were stained with Hematoxylin and Eosin (Scale bar: 200 μm). Sections at higher magnification (Scale bar: 50 μm.). Experiments were repeated four times independently with similar results. (f). Healthy mice were administered with DSPE-PCB micelle, saline, Polysorbate 80, or sodium decanoate through oral gavage twice daily for 14 consecutive days. On day 0 and 14, before and 1 h after the oral administration, blood serum was collected and the endotoxin and pro-inflammatory cytokines were measured with the mouse magnetic Luminex® assays (N=4 biologically independent samples, mean ± SD). A one-way analysis of variance with Tukey multi-comparison was used for statistical analysis; compare with saline within the same group.