Objective Eye drop is the currently main formulation for ocular drug delivery on the market.However, the efficiency of this formulation is often limited by serious drug loss from the ocular surface, poor penetration across the cornea and reduced residence time due to tear excretion and clearance.To overcome these shortcomings, nanoparticles, especially cubic liquid crystalline nanoparticle (cubosome) was preferred due to its inherent advantages.In this study, the timolol maleate loaded cubosomes were fabricated and characterized.Methods GMO and Poloxamer 407 (9∶1, w/w) were used to prepare cubosomes utilizing high-pressure homogenization.The particle size, PDI and zeta potential of the prepared cubosomes were measured by nano zetasizer.The encapsulation efficiency was measured by ultracentrifugation.Polarized light microscopy and small angle X-ray scattering (SAXS) were utilized to determine the specific crystalline form of cubosomes.In vitro drug release was performed using an equilibrium dialysis method.Experiment in rabbit model of glaucoma was conducted to compare pharmacodynamics of timolol cubosomes and timolol solution by measuring the decrease of intraocular pressure.Results and discussion The cubosomes showed an average particle size of 180nm and an average zeta potential of-15mV.The encapsulation efficiency was over 85%, suggesting that most of the timolol maleate was entrapped in the cubosomes.Drug release profile showed timolol maleate cubosomes could attain 5h sustained release behavior.Animal experiment indicated better intraocular pressure lowering effect oftimolol cubosomes.Conclusion The timolol maleate cubosomes showed good physicochemical stability and high encapsulation efficiency.Moreover, it exerted better intraocular pressure lowering effect, which showed great potential in treating glaucoma.