The Trans-corneal Barrier Evaluation Of Dexamethasone-loaded Lipid Nanoparticles And Lipid Nanoparticles-thermosensitive Gel

Dexamethasone is one of the effective corticosteroids to treat uveitis clinically.Due to the limited bioavailability of dexamethasone at aqueous humor,it requires the long-term and frequent administration of dexamethasone to maintain its pharmaceutical effects.However,the long-term and frequent administration causes the dexamethasone accumulation of non-target sites,thus might induce iatrogenic diseases and symptoms.One of the effective methods to enhance the bioavailability of dexamethasone at aqueous humor is to reinforce corneal penetration of dexamethasone.In this study,the trans-corneal ability of lipid nanoparticles and lipid nanoparticles-thermosensitive gel was evaluated.Moreover,these two delivery systems were screened based on their bioavailability at aqueous humor.Lipid nanoparticles were prepared by high pressure homogenization.Lipid nanoparticles were white.The particle size（D90）of lipid nanoparticles was 333.7±10.61 nm,and the zeta potential of lipid nanoparticles was–（28.03±0.95）m V.The entrapment efficiency of lipid nanoparticles was（64.32±1.89）%.Both poloxamer 407 and 188 were dissolved to prepare poloxamer-based thermosensitive gel via cold solution technique.Then gel was mixed with lipid nanoparticles evenly to form lipid nanoparticles-thermosensitive gel.The gelation temperature of lipid nanoparticles-thermosensitive gel was 32.7±0.3℃and was detected by tube inversion method.The viscosity of lipid nanoparticles-thermosensitive gel was 132.5±6.85 m Pa·s at 25℃and 35830±785.4m Pa·s at 33℃respectively.Viscosity was determined by a rotational viscometer with a temperature-controlled device.The p H values of both lipid nanoparticles and lipid nanoparticles-thermosensitive gel were6.54±0.06 and 6.38±0.10 respectively.Their osmolarities were 278.3±3.06and 261.7±7.09 mosmol·kg^-1respectively.Both their p H values and osmolarities were all met the ocular preparation requirement.The contact angles of lipid nanoparticles and lipid nanoparticles-thermosensitive gel were（39.8±2.69）°and（38.6±3.24）°respectively via using a contact angle detector.Both of their contact angles were less than 60°,indicating that both lipid nanoparticles and lipid nanoparticles-thermosensitive gel can spread on corneal surface.The in vitro drug release of both lipid nanoparticles and lipid nanoparticles-thermosensitive gel were tested through dialysis.The drug release velocity of both lipid nanoparticles and lipid nanoparticles-thermosensitive gel was slower than that of Tobra Dex（commercial eyedrop）.Lipid nanoparticles-thermosensitive gel has stronger sustained release than lipid nanoparticles.There was no burst release happening in the drug release of lipid nanoparticles-thermosensitive gel.Data of lipid nanoparticles,lipid nanoparticles-thermosensitive gel and Tobra Dex were fitted to different release kinetic models:Zero Order,First Order,Higuchi,Ritger Peppas and Hixson Crowell.The drug release of lipid nanoparticles and Tobra Dex belonged to Fick diffusion,while the drug release of lipid nanoparticles-thermosensitive gel involved Fick diffusion and skeleton erosion.The in vitro corneal permeation of both lipid nanoparticles and lipid nanoparticles-thermosensitive gel were tested through Franz diffusion sets.The cumulative corneal permeation per unit area of lipid nanoparticles was1.52±0.13-fold higher than that of lipid nanoparticles-thermosensitive gel at 12 h.The of lipid nanoparticles was 1.49±0.20-fold greater than that of lipid nanoparticles-thermosensitive gel.The matrix of thermosensitive gel impedes drug diffusion,thus limits the in vitro corneal permeation of lipid nanoparticles-thermosensitive gel.The epithelial cellular barrier permeation of both lipid nanoparticles and lipid nanoparticles-thermosensitive gel were tested through self-built MDCK epithelial cellular barriers.The cumulative permeation of lipid nanoparticles was 1.85±0.29-fold greater than that of lipid nanoparticles-thermosensitive gel at 1.5 h and was similar with that of lipid nanoparticles-thermosensitive gel at 3.0 h.The epithelial permeation of lipid nanoparticles-thermosensitive gel processed slowly,which might cause the less in vitro corneal permeation than lipid nanoparticles did.In order to evaluate in vivo corneal permeation of both lipid nanoparticles and lipid nanoparticles-thermosensitive gel,the pharmacokinetics of tear clearance and aqueous humor have been developed.Based on the results of pharmacokinetics of tear clearance,the of lipid nanoparticles-thermosensitive gel was 6.23±1.26-fold higher than that of Tobra Dex,and the of lipid nanoparticles was1.96±0.36-fold higher than that of Tobra Dex.As for pharmacokinetics of aqueous humor,the of lipid nanoparticles-thermosensitive gel and lipid nanoparticles was 2.23±0.41-fold and 1.39±0.19-fold greater than that of Tobra Dex.The of lipid nanoparticles-thermosensitive gel was2.78±0.30 h,which was the longest among all the experimental groups.Primarily,lipid nanoparticles-thermosensitive gel performed better dexamethasone retention at precorneal region than lipid nanoparticles did.Thus,lipid nanoparticles-thermosensitive gel can enhance in vivo corneal penetration and in vivo retention of dexamethasone at aqueous humor more significantly.After multiple administration of lipid nanoparticles and lipid nanoparticles-thermosensitive gel,the ocular irritation test showed that there was no sign of any irritation on rabbit ocular surface after 7-day administration period.Tissues（Cornea,iris and retina）were sliced after the solidification of melted paraffin.Later,they were all stained by hematoxylin and eosin,and examined microscopically.After multiple administration of lipid nanoparticles and lipid nanoparticles-thermosensitive gel,tissues remained their normally biological structure,and cells within these tissues were in tight junctions and clear shapes.The HCE-T cellular uptake of lipid nanoparticles and lipid nanoparticles-thermosensitive gel was studied by flow cytometry.The cellular relative fluorescent intensity of lipid nanoparticles was 2.28±0.30-fold and 2.20±0.53-fold greater than that of lipid nanoparticles-thermosensitive gel at 0.5 h and 1.0 h respectively.Until 2.0 h,the corneal epithelial cellular uptake of lipid nanoparticles was larger than that of lipid nanoparticles-thermosensitive gel.The cellular uptake mechanism of lipid nanoparticles and lipid nanoparticles-thermosensitive gel was studied through the combination of endocytosis inhibitors and flow cytometry.Corneal epithelial cells internalized lipid nanoparticles through pinocytosis.As for lipid nanoparticles-thermosensitive gel,cells internalized them via pinocytosis,clathrin-mediated endocytosis and lipid raft/caveolin-mediated endocytosis.In terms of drug release,ex vivo/in vivo corneal permeation,in vivo pre-corneal retention and cellular uptake,lipid nanoparticles-thermosensitive gel can reduce the tear clearance of dexamethasone and prolong the retention of dexamethasone at pre-corneal region more effectively than lipid nanoparticles.Therefore,lipid nanoparticles-thermosensitive gel can accumulate more dexamethasone at cornea to form a reservoir and reinforce concentration of dexamethasone and prolong the retention of dexamethasone at aqueous humor.Overall,lipid nanoparticles-thermosensitive gel displays its potential to enhance the bioavailability of dexamethasone at aqueous humor.