Retinal vascular permeability assay

Retinal vascular permeability was examined by fluorescein angiography and the Evans Blue (EB; Sigma-Aldrich) assay. Briefly, rhVEGF and/or Apa-HSA-PEG nanoparticles prepared in 1 µL PBS were injected into the vitreous cavity of one eye of each mouse, with an equal volume of PBS injected into the contralateral eye. For angiography, FITC-dextran (MW 40,000 Da; Sigma-Aldrich) was injected intravenously, and the mice were sacrificed 30 minutes later. The eyes were enucleated and fixed in 10% formalin (Sigma-Aldrich). The retinas were then prepared as flat mounts on glass slides and viewed with a fluorescence microscope (Nikon, Melville, NY, USA) in four randomly selected areas of each retina as described previously.10 For quantification of vascular leakage, 150 µL of EB (20 mg/mL in PBS, sonicated and filtered) was injected intravenously, and the mice were sacrificed 4 hours later. Two hundred microliters of blood was obtained, and the retinas were collected, dried, and weighed. EB was extracted from the dried retinas with formamide (Sigma-Aldrich) at 78°C overnight. The absorbance of the EB dye in blood and retinal tissues was measured at 620 and 740 nm using a spectrophotometer (BioTek Instruments). The amount of EB dye was calculated from a standard curve of EB in formamide. EB permeation was calculated as follows: [retinal EB amount/retinal weight]/[blood EB concentration × circulation time]. EB permeation of treated eyes was normalized relative to that of each contralateral PBS control.