Fig. 3: Poly(CEP) suppresses RPE cell proliferation and migration in a concentration-dependent manner.

a Proliferation in RPE cells exposed to poly(CEP) micelles at 0.1, 0.5, and 1 wt% with TNT were ascertained by Ki67 IF. Only 1 wt% poly(CEP) visibly reduced the number of Ki67 positive nuclei. Membranes are indicated by white arrows. b Proliferation of RPE cells exposed to five concentrations (below CMC: 0.001 and 0.01 wt%; above CMC: 0.1, 0.5, and 1 wt%) of poly(CEP) without TNT were immunostained with Ki67. Nuclei was stained in blue with Hoechst stain. Scale bar for a, b, 100 µm. c All five concentrations in (b) and media only control from (a) were quantified for co-localisation of Ki67 with nuclei (using Hoechst stain). This co-localisation was plotted as the Ki67 positive nuclei (%) indicating actively proliferating cells at the endpoint of 72 h. Only poly(CEP) ≥ 0.5 wt% showed significant inhibition of proliferation compared to media only control. Data represents mean ± s.d. of five replicates. d Cell proliferation was monitored by plotting a curve using the mean area confluence (%) of cells imaged at regular intervals (10–12 h) exposed to five concentrations of poly(CEP) alone. Only poly(CEP) at ≥ 0.5 wt% showed significant suppression of proliferation compared to control (Media only). Data represents mean ± s.d. of three replicates. e Cytocompatibility of poly(CEP) at the same concentrations was studied using cytotoxicity lactate dehydrogenase (LDH) assay. LDH release (%) was negligible at all concentrations of poly(CEP) and media only control. Lysis buffer (provided by the manufacturer) was used as a positive control. Data represents mean ± s.d. of three replicates. f Scratch-wound healing assay was conducted to study RPE cell migration. Cells were exposed to two concentrations of poly(CEP) (0.1 and 1 wt%), with or without TNT. Phase contrast images of all groups were captured at four time points (0, 12, 24, and 36 h) with scratch-wound mask in blue. Scale bar, 300 µm. g Wound recovery (%) was quantified at three time-points (12, 24, and 36 h) post-scratch. Only poly(CEP) at 1 wt% displayed significant attenuation of migration compared to media only control at the endpoint of 36 h. This attenuation was also observed for 1 wt% poly(CEP) + TNT compared to TNT. Data represents mean ± s.d. of five replicates. Statistical analyses were performed for c–e and g using one-way ANOVA, followed by Tukey’s honest significance difference (HSD) post hoc test. ns, not significant.