Biodegradable Microspheres with Long-term Release of Survival Factors in the DBA/2J Mouse Model of Glaucoma

Abstract

Abstract: : Purpose: Neurotrophic growth factors have been shown to enhance retinal ganglion cell (RGC) survival when given as intravitreous bolus injections. Microspheres fabricated from biodegradable polymers can allow for a more gradual, physiological release of these factors. The DBA/2J mice have been shown to develop pigment dispersion glaucoma with symptoms beginning as early as four months. These mice provide a suitable model for testing glaucoma therapeutics. We wish to promote RGC survival in the DBA/2J mice using biodegradable polymers releasing retinal survival factors. Methods: Microspheres with GDNF, CNTF, and BDNF were formed using a spontaneous emulsification technique in which a solution of the polymer, AOT, and drug of interest were vortexed then added to a rapidly stirring solution of polyvinyl alcohol in water to precipitate the polymer/AOT/drug solution forming poly(lactic-co-glycolic acid) (PLGA) spheres of controlled size. Rhodamine labeled microspheres were created for in vivo imaging and histological analysis. Release studies were performed in vitro in PBS for 60 days and release was quantified using ELISA assays for the individual growth factors. Two-month-old DBA/2J mice were anesthetized and injected with 1 µl of concentrated microspheres (2% rhodamine microspheres) into the vitreous. Evaluation was performed by in vivo imaging using a flourescent microscope and by histological sections. Results: ELISA assays showed an insignificant initial burst of 2 to 4 ng of growth factor per mg, which plateaued for ~25 days, followed by a phase of gradual release lasting approximately 30 days. Release curves differed significantly depending on microsphere size and the survival factor in question. In vivo flourescent images show rhodamine-labeled microspheres immediately post-operatively and 7 days afterwards. Histological sections demonstrate microspheres in the vitreous cavity. No signs of uveitis or inflammation were noted. Discussion: We have fabricated and characterized microspheres to deliver GDNF and other factors in a controlled manner for up to 60 days with minimal initial burst. The DBA/2J mouse tolerates injections of these polymers, and it is a promising model for studying such therapeutics.

Keywords: neuroprotection • growth factors/growth factor receptors