Fig 6. Effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on membrane stability.

(A) Modification of hRBC with NHS-pDMAA-Cy3 polymers does not induce membrane destabilization as evidenced by the absence of PS-Annexin V signal under physiological conditions. Both unmodified and modified (100 μM polymer solution) hRBC were incubated in 1X PBS for one hour at 37°C. Annexin V-Alexa488 binding to externalized phosphatidylserine was used to monitor membrane destabilization and oxidative damage (green fluorescence). Cy3-modification demonstrated by red fluorescence. Epifluorescent images of PS-Annexin V and Cy3 channels captured under oil at 60x using an inverted Leica microscope. Individual channels were merged using Image J. Scale bars measure 50 μm. Images were background corrected and the brightness/contrast for each channel was balanced using Image J software. (B) Effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on membrane damage during oxidant exposure. Modification of hRBC with NHS-pDMAA-Cy3 polymers may protect against membrane damage under oxidizing conditions demonstrated by a potential mitigation of phosphatidylserine-Annexin V signal. Both unmodified and modified (100 μM polymer solution) hRBC were incubated in 0.2 mM CuSO₄/2.5 mM ascorbate solution for one hour at 37°C. Annexin V-Alexa488 binding to externalized phosphatidylserine was used to monitor membrane destabilization and oxidative damage (green fluorescence). Cy3-modification demonstrated by red fluorescence. Epifluorescent images of PS-Annexin V and Cy3 channels captured under oil at 60X using an inverted Leica microscope. Individual channels were merged using Image J. Scale bars measure 50 μm. Images were background corrected and the brightness/contrast for each channel was balanced using Image J software. (C-F). Time point analysis of the effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on membrane damage during oxidant exposure. Modification of hRBC with NHS-pDMAA-Cy3 polymers may protect against membrane damage under oxidizing conditions demonstrated by a potential mitigation of phosphatidylserine-Annexin V signal over time. To further investigate the temporal regulation of these events, both unmodified and modified (100 μM polymer solution) hRBC were incubated in 0.2 mM CuSO₄/2.5 mM ascorbate solution for 10 (C), 30 (D), 60 (E), or 120 (F) minutes at 37°C. Annexin V-Alexa488 binding to externalized phosphatidylserine was used to monitor membrane destabilization and oxidative damage (green fluorescence). Cy3-modification demonstrated by red fluorescence. Epifluorescent images of PS-Annexin V and Cy3 channels captured under oil at 60X using an inverted Leica microscope. Individual channels were merged using Image J. Scale bars measure 50 μm. Images were background corrected and the brightness/contrast for each channel was balanced using Image J software.