VITAMIN E INCREASES THE BIOACTIVITY IN RETINAL CELLS OF MICROENCAPSULATED GLIAL CELL LINE DERIVED NEUROTROPHIC FACTOR

Abstract

Purpose: To evaluate the bioactivity of GDNF released form PLGA microspheres (MS) in retinal cells in presence of vitamin E (vit-E) as antioxidant.

Methods: GDNF-loaded PLGA MS were prepared by S/O/W emulsion/solvent-extraction-evaporation method. 20µg of recombinant human GDNF were suspended in the inner-phase of the emulsion composed by PLGA/CH2Cl2 solution (MS-A) or PLGA/CH2Cl2 solution including 20µl of vit-E (MS-B). MS were characterized in terms of production yield, SEM, mean-particle-size and particle-size-distribution, encapsulation efficiency (ELISA) and in-vitro release studies. For bioassays, retinal from post-natal day-10 (B6 mice) were isolated and exposed to conditioned media released from MS-A and MS-B. At 40h post plating, the percentage of death cells was analysed (TUNEL).

Results: The microencapsulation method used led to production yield of 80% for both formulations. MS (20-40µm) resulted spherical with smooth surfaces and absence of pores for MS-A but with small surface pores in MS-B. The inclusion of vit-E in MS-B increased the protein entrapment from 23.4±0.5% to 30.4±1.1%. A three-phasic sustained GDNF in-vitro release was obtained for both formulations for 133 days. In phase-I 37 and 33 pgGDNF/mgMS/day were obtained for MS-A and MS-B respectively. In phase-II 144 pgGDNF/mgMS/day for MS-A and 187 pgGDNF/mgMS/day for MS-B were denoted. Finally, 8 (MS-A) and 14 (MS-B) pgGDNF/mgMS/day were observed. Less retinal cell death (p<0.001) was observed when cultured in presence of GDNF released from MS-B (8.2%) in comparison to MS-A (15.9%).

Conclusions: Inclusion of antioxidants is a useful strategy to increase the biological neuroprotective activity of microencapsulated neurotrophic factors such as GDNF.

Keywords: 449 cell survival • 615 neuroprotection • 424 antioxidants