Sterilized Gdnf Microspheres For Intraocular Administration. Influence Of Vitamin E Addition

Abstract

Purpose: : Intravitreal injection of Glial-cell-line-Derived Neurotrophic Factor (GDNF) is an interesting strategy in the treatment of optic nerve neurodegenerative diseases. The objective of the present work was the characterization of sterilized PLGA microspheres (MSs) loaded with GDNF, with and without Vit E for the intravitreal administration as a new strategy of neuroprotective therapies.

Methods: : MSs were prepared by the S/O/W emulsion-solvent evaporation method employing 20µg of GDNF and 20µl (Formulation A) or 0µl (Formulation B) of Vit E. The formulations were sterilized using ⁶⁰Co as the radiation source in the γ-radiation unit at Aragogamma S.L. (Barcelona, Spain). A γ-radiation dose of 25 kGy was employed to ensure effective sterilization. Morphological and particle size characterization were performed by scanning electronic microscopy (SEM) and Microtrac S3500 respectively. In vitro release studies were carried out for 19 weeks by suspending the MSs in 1.5ml of a release media composed by PBS (pH 7.4), 1% BSA and 0.02% Sodium Azide. At pre-set times (1h, 24h and once a week for 19 weeks) the release medium was collected and GDNF levels quantified using ELISA. Bioassays in retinal ganglion cells (RGC) were performed to evaluate the functionality of GDNF/Vit E-PLGA MSs.

Results: : MS size distribution was in the 20-40µm range. The encapsulation efficiency was different in both formulations, 19.0±0.8ngGDNF/mgMSs (Formulation A) and 8.7±0.6ngGDNF/mgMSs (Formulation B). The initial burst effect (24h release assay) was observed with values of 14.1±0.4ngGDNF/mgMSs for formulation A and 6.2±0.8ngGDNF/mgMS for formulation B. Following initial burst release, 35.8±8.6pgGDNF/mgMSs/day (Formulation A) and 35.9±6.6pgGDNF/mgMSs/day (Formulation B) was detected for the first 9 weeks of release test. From weeks 9 to 19 of the assay, GDNF released from MSs of Formulation A and B were determined to be 6.0±1.1pg/mgMPs/day and 3.7±0.8pg/mgMPs/day respectively. A significant increase in RGC was observed in cultures treated with GDNF/Vit E-PLGA MSs as compared to control (MSs loaded with 20µl Vit E).

Conclusions: : γ-radiation is an appropriate method to final sterilization of GDNF/Vit E-PLGA MSs prior to intravitreal administration. The inclusion of Vit E improved the GDNF encapsulation efficiency and modulated the in vitro release of the neurotrophic factor.

Keywords: neuroprotection • retina • optic nerve