PURPOSE: The main goal of this study was to encapsulate Pioglitazone (PGZ), in biodegradable polymeric nanoparticles as a new strategy for the treatment of ocular inflammatory processes. METHODS: To improve their biopharmaceutical profile for the treatment of ocular inflammatory disorders, nanospheres (NSs) of PGZ were formulated by factorial design with poly (lactic-co-glycolic acid) polyethylene glycol (PLGA-PEG). Interactions drug-polymer have been carried out by spectroscopic (X-ray spectroscopy, FTIR) and thermal methods (DSC). The PGZ-NSs were tested for their in vitro release profile, cytotoxicity, and ocular tolerance (HET-CAM® test); ex vivo corneal permeation, and in vivo inflammatory prevention and bioavailability. RESULTS: The optimized system showed a negative surface charge of -13.9 mV, an average particle size (Zav) of around 160 nm, a polydispersity index (PI) below 0.1, and a high encapsulation efficiency (EE) of around 92%. According to the DSC results, the drug was incorporated into the NSs polymeric matrix. The drug release was sustained for up to 14 h. PGZ-NSs up to 10 μg/ml exhibited no retinoblastoma cell toxicity. The ex vivo corneal and scleral permeation profiles of PGZ-NSs showed that retention and permeation through the sclera were higher than through the cornea. Ocular tolerance in vitro and in vivo demonstrated the non-irritant character of the formulation. CONCLUSION: The in vivo anti-inflammatory efficacy of developed PGZ-NSs indicates this colloidal system could constitute a new approach to prevent ocular inflammation. KEYWORDS: PLGA-PEG; drug delivery; nanospheres; ocular anti-inflammatory efficacy; pioglitazone