Ranibizumab, Bevacizumab, and Aflibercept accumulation and their effect on cell migration and permeability on human ARPE-19 cells
Purpose : To evaluate intracellular accumulation and the effect of bevacizumab, ranibizumab and aflibercept on cellular migration and permeability in a human retinal pigmented epithelium (RPE) cell line.
Methods : Experiments were performed on ARPE-19 cells and anti-VEGF drugs were diluted to a concentration equivalent to their clinical doses. Anti-VEGFs were labeled with Alexa 488 fluorochrome to detect intracellular accumulation by flow cytometry at 1 hour, 1 day and five days. Further, transepithelial electrical resistance (TEER) was measured in transwells at 2, 4, 6, 12 and 24 hours to assess the effect of anti-VEGFs on RPE permeability. Moreover, TEER was also measured at the same time points in the presence of different doses of H2O2 to replicate the oxidative environment observed in Age-related Macular Degeneration (AMD). Wound healing was assessed to determine the effect of the drugs on cellular migration during 72 hours to measured cell covered area by ImageJ software.
Results : The three studied drugs were observed to accumulate inside the cells and they were still detectable five days after being added (p<0.001). A dose-dependent increased in cell permeability was observed in cells treated with H2O2 (p<0.05) that was reverted from the time point of 12 hours and became non-significant. Anti-VEGF drugs did not affect the permeability along time and they were able to reduce the effect of H2O2. Cells treated with anti-VEGF drugs at the beginning of the experiment showed a significant decrease in the damage produced by H2O2 at 4, 6 and 12 hours (p<0.05) with no significant difference between the treatments. On the contrary, when anti-VEGF treatment was used 6 hours after the beginning of the experiment, none of the 3 drugs decreased the deleterious effect of H2O2 in TEER. Anti-VEGF drugs did not affect cellular migration.
Conclusions : Intracellular accumulation of bevacizumab, ranibizumab and aflibercept does not seem to be toxic or affect cell permeability and migration. Moreover, our study suggests that anti-VEGFs have a positive effect on the barrier function of the RPE.