In vivo biodistribution

Animals were housed and treated according to the Louisiana State University animal care policies and in compliance with the American Veterinary Medical Association (AVMA). The experimental protocols were approved by Louisiana State University Institution Animal Care and Use Committee (IACUC).

Adult (37–46 weeks old) Wistar rats (Envigo, Huntingdon, United Kingdom) were divided randomly into four groups: three experimental groups which received the same dose of fluorescent nanoparticles but were euthanized at different times (15, 30, and 60 min after application), and one control group which received PBS (control). The treatment was prepared by suspending dry nanoparticles in PBS at 20 mg/mL. One drop of either the nanoparticle suspension or PBS, approximately 12 µL, was applied to both eyes of each animal. Controls and treated animals were kept separate during the incubation period to prevent cross contamination of treatments between animals. At different time points tested, animals were euthanized in a CO₂ gas chamber. The eyes were immediately enucleated after euthanasia, immersed in OCT freezing media, and snap frozen in isopentane cooled by liquid nitrogen. Eyes were cryo-sectioned (Thermo Shandon Cryotome E, Cambridge, UK) at 8 μm and imaged using an Eclipse Ti2 microscope (Nikon Instruments Inc., Melville, NY, USA). Both phase-contrast and fluorescence images were taken to capture tissue morphologies and fluorescence from nanoparticles.