| Objective To prepare econazole nitrate nanoparticles (ECZ-RS100-NP) eye drops by uniform design and determine their size, charge, encapsulation efficiency and drug loading and other basic properties; To detect econazole concentration changes in aqueous humor and cornea after its application in rabbit eyes and analyze the pharmacokinetics.Methods We prepared single-factor condition primarily and examined the ECZ absorbance in natural light, darkness, exposed in air and vacuum drying in 60℃for 5 hours, then analyzed the nanoparticles stability in different preparation conditions. We compared the effect on size and charge of nanoparticles with purified water and diluted PBS buffer after ultrafiltrated, studied the changes in nanoparticles size and charge under added different agents such as sodium hyaluronate, polyvinyl alcohol, glycerin and polyethylene glycol, respectively. The appropriate pH value was determined after nanoparticles size and charge in pH value between 5.00 and 7.50 were investigated. In the 8 levels of 6 factors, we developed uniform design table and summed up the test results scores, then we had a multiple linear regression equation and determined the preparation conditions and described the nanoparticles on morphology, TEM photographs, size, charge, encapsulation efficiency and drug loading. Japanese White Rabbits with free eye disease were randomly divided into nanoparticles group (as Experiment group) and suspension group (as Control group), 0.3% ECZ nanoparticles eye drops was used as a test drug and 0.3% ECZ suspension (ECZ-SP) eye drops was used as a control drug. In the single-dose group, 50μl of test drug or control drug was dropped on rabbit eyes. In the loading-dose group, 50μl test drug or control drug was dropped on rabbit eyes for 3 times with an interval of 3min. After the last time application, at the time point such as 5min,15min,30min,45min,60min,90min,120min,180min,240min and 300min, aqueous humor was drawn and stored in -60℃and the cornea was dissected and weighed after the rabbit was killeded, respectively. After samples were processed and abstracted, the drug was detected using reversed phase high performance liquid chromatography (RP-HPLC). The ECZ concentrations in aqueous humor and cornea of rabbits at different time points were compared. The pharmacokinetics parameters were conducted using 3p97 pharmacokinetics software.Results ECZ-RS100-NP eye drops showed light blue opalescent appearance and electron microscopy images showed uniform particle dispersion. The particle size was 50.47nm and charge was +31.04 mV. Encapsulation efficiency was 87.63% and drug loading was 25.18%. The drug levels were higher than suspension eye drops, and there were significant differences between two groups.(P <0.01) for the aqueous humor at5min time point, and for cornea in a single dose, at 5min, 15min, 30min, 45min, 60min, 90min and 120min time points. In loading-dose group, drug levels were higher than suspension eye drops, and the comparison between the two groups showed significant difference (P <0.01 or P <0.05) for the aqueous humor at 5 min, 15 min, 30 min, 45 min, 120 min time points, and for corneas at time points of 5min, 15min, 30min.In the single dose group, the drug peak time (Tmax) in aqueous humor for experimental group and control group were 5min and 60min, respectively. The half-lives of the drug (t1/2) in the aqueous humor for experimental group and control group were 47.38min and 51.95min. The drug concentration-time area under the curve (AUC) during the 0-300min in aqueous humor were 10.76 (μg/ml )·min and 86.47 (μg/ml)·min, respectively. In the loading-dose group, the Tmax in the aqueous humor of experimental group and control group were both 45min, respectively. The t1/2in the aqueous humor for experimental group and control group were 91.54min and 150.35min, The AUC0-300min in the aqueous humor were 87.89 (μg / ml)·min and 241.74 (μg/ml)·min, respectively.For the single dose group, t1/2 in the cornea for experimental group and control group were 44.68min and 30.55min, respectively. The AUC0-300 were 555.48 (μg /mg)·min and 830.96 (μg/mg)·min, respectively. For loading-dose group, the t1/2 in cornea of experimental group and control group were 58.26min and 57.79min, respectively, The AUC0-300 were 2162.13(μg/mg)·min and 2446.27 (μg/mg)·min, respectively.Conclusion The nanoparticels eye drops obtained has smaller particle size, high zeta potential and ideal encapsulation efficiency and drug loading. Compared with the control group, ECZ concentration in cornea in experimental group was higher for the single-dose group. The drug levels in aqueous humor in experiment group before 45 min were higher than the control group for the loading-dose group. The ECZ nanoparticle eye drops will be potential to treat fungal keratitis in the future. |
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