Intraocular distribution and kinetics of intravitreally injected non-biodegradable nanoparticles in rabbits

Abstract

Introduction: Recently, intravitreal injection of large protein drugs has been used for the treatment of many retinal diseases. Although molecular weight is the main factor in determining the kinetics, the exact mechanisms underlying drug delivery and intraocular distribution and elimination of these drugs has not been elucidated. Efforts have been made to enhance drug delivery to ocular tissues using various kinds of drug delivery systems, including nanoparticles. This study was performed to analyze the intraocular distribution and kinetics of intravitreally injected non-biodegradable nanoparticles in rabbit eyes, to help understand the intraocular biodistribution of intravitreous materials and drugs. Methods: Four kinds of fluorescent non-biodegradable polystyrene nanoparticles were used in this study: 25 nm and 250 nm diameter nanoparticles to investigate the intraocular distribution and elimination pathways of nanoparticles and intraocular kinetics of nanoparticles, and 50 nm and 200 nm diameter nanoparticles to investigate the intraocular kinetics of nanoparticles. A 0.034 ml mixture of 25 nm nanoparticles and 250 nm nanoparticles (1:1) was injected intravitreally into 24 eyes of 12 New Zealand white rabbits. Four eyes were enucleated at each time point (1 hour, 1 day, 7 days, 14 days, 21 days, and 30 days after the injection) and immediately frozen at -80 °C. Fluorescence microscopic imaging was performed on one eye at each time point. Fluorescence microscopic imaging of the cross section of each frozen enucleated eyeball was performed using a custom-built laser-scanning confocal system modified for wide-field imaging before it began to melt. Intraocular distribution of nanoparticles was analyzed serially with software at each time point. The other three eyes from each time point were used for fluorescence measurements. A 10-fold diluted solution of 50 nm nanoparticles was injected intravitreally into 15 right eyes of 15 New Zealand white rabbits and a 100-fold diluted solution of 200 nm nanoparticles was injected intravitreally into 15 right eyes of 15 New Zealand white rabbits. Three eyes per solution group per time point were enucleated for fluorescence measurements at 1 hour, 1 day, 7 days, 14 days, and 30 days after the injection and immediately frozen at -80 °C. The optical density of fluorescence was determined using a fluorescence microplate reader after the separation of frozen vitreous, aqueous humor, and retina, and the concentration of nanoparticles in each sample was determined. Results: Serial imaging of the intraocular distribution of nanoparticles showed that the intensity of fluorescence did not decrease in the vitreous over time and the injected nanoparticles moved posteriorly to the retina from 7 days after the injection. However, the nanoparticles could not penetrate into the deeper retinal structures and accumulated on the internal limiting membrane. Furthermore, the nanoparticles moved anteriorly in the vitreous at 1 hour and 1 day post-injection, and the fluorescence increased in the anterior chamber until 14 days after the injection and it decreased thereafter. In the image of the eye that had been enucleated 21 days after the injection, we found that the 25 nm nanoparticles moved across the ciliary body into the choroid and episcleral space. Overall, the concentration of nanoparticles in the vitreous did not change at all the measured time points, irrespective of sizes. In the aqueous humor, the concentration of 50 nm and 200 nm nanoparticles did not increase after the 1-day time point and the 25 nm and 250 nm nanoparticles did not increase after the 7-day time point. Nanoparticles were detected in the retinal tissue. Conclusion: The result of the intraocular distribution, changes in concentration, and elimination pathways of various-sized non-biodegradable nanoparticles may be used as basic data in the studies of intraocular drug delivery and pharmacokinetics using nanoparticles in the future. It might also indicate the possible mechanisms underlying intraocular elimination and movement of intravitreally injected drugs, which are currently used to treat retinal diseases.