| Purpose:The objective of the present study was to investigate the cou-6 flexible nanoliposomes containing dipotassium glycyrrhizinate regarding in vitro cellular uptake and in vivo corneal permeation and the possible mechanics, to provide theoretical basis and research foundation for the use of flexible nanoliposomes as a new drug delivery system in ocular drug delivery.Methods : 1. Preparation of cou-6 nanoliposomes and flexible nanoliposomes(containing dipotassium glycyrrhizinate) and the research of their physical and chemical properties: They were prepared by the film dispersing method. The entrapment efficiency of cou-6-loaded liposomes was determined using a high-performance liquid chromatographic method. To observe the morphological characterization of the two nanoliposomes by transmission electron microscop. The mean particle size, polydispersity and the zeta potential of the nanoliposomes was determined using a particle size analyzer. The elasticity of the two nanoliposomes was measured by the film extrusion method. At last, compared their physical and chemical indicators.2. Safety evaluation in vivo and in vitro: We tested the cytotoxicity of the dipotassium glycyrrhizinate, nanoliposomes and flexible nanoliposomes on HCECs with standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) testing. benzalkonium bromide(a preservative widely used in ophthalmic solutions) was also tested as a control.3. In vitro HCECs uptake and mechanical characters: After the cells were grown to confluence, the cou-6 solution, cou-6 nanoliposomes and flexible nanoliposomes were added respectively and tested the cell uptake at each time point. The cells were pre-incubated with the different inhibitors or at 4℃ to test the cell uptake mechanism of two nanoliposomes. We also analysised and compared the mechanical similarities and differences between two nanoliposomes by confocal laser scanning microscopy.4. In vivo corneal permeation testing: The concentrations of cou-6 in the aqueous humor and corneas were tested at each time point after topical administration with rabbits and mice as experimental animals and the fluorescence of cou-6 in the cornea was observed by confocal laser scanning microscopy to analysis and compare the differences of corneal uptake between two nanoliposomes after ocular administration.5. The anti-inflammatory activity of dipotassium glycyrrhizinate and flexible nanoliposomes: The eye local inflammatory models were set up by instillating arachidonic acid solution in rabbit eyes to evaluate the anti-inflammatory activity of dipotassium glycyrrhizinate and flexible nanoliposomes.Results : 1. Both the nanoliposomes and flexible nanoliposomes had high encapsulation efficiency. The particle appearance observation showed that both of the two liposomes were uniform circular with conformable particle size and consistent dispersion coefficient, but the zeta potential was higher after being modified by dipotassium glycyrrhizinate. Under high magnification(x150K) by transmission electron microscop, the flexible nanoliposomes showed shrinkage rims but this observation was not observed to the nanoliposomes, and their rims were smooth. The phenomenon hinted that the lipid bilayer of flexible nanoliposomes had some elasticity. The elasticity evaluation confirmed that the flexible nanoliposomes had increased elasticity.2. Dipotassium glycyrrhizinate showed certain cytotoxicity on HCECs only in high concentration or after a long-time cell incubation, and the cytotoxicity was time and concentration dependent. After 1h of incubation, no obvious cytotoxicity was detected for either the liposomal or flexible liposomal formulation.3. In vitro HCECs uptake testing: The cell uptake reached equilibration at 5 min in the cou-6 solution and liposomal formulation, and the level of cou-6 uptake in the liposomal formulation was significantly greater than that in free cou-6 solution. The uptake of the flexible liposomal formulation was time dependent. In the flexible liposomal formulation, longer incubation induced significantly more cou-6 uptake from 5 min to 60 min and the level of cou-6 uptake was greater than that in liposomal formulation at every time point.In vitro energy-dependent or-independent test: The cellular uptake of nanoliposomes at 4℃ or in the presence of sodium azide at 37℃ for 30 min significantly decreased by 20.47% and 23.10%, respectively, while the cellular uptake of flexible nanoliposomes was not decreased apparently. This phenomenon could be regarded as an index of energy-dependent uptake of nanoliposomes.After pre-incubation with different inhibitors of endocytosis, we found that the inhibitory effect of chlorpromazine on the cellular uptake of nanoliposomes was most obvious, following by hypertonic sucrose and MβCD. However, that of MβCD on the cellular uptake of flexible nanoliposomes was most obvious while the effect of hypertonic sucrose was less than that on the cellular uptake of nanoliposomes.When HCECs were incubated with nanoliposomes, punctuated fluorescence was observed by confocal laser scanning microscopy in their cytoplasm, while in flexible nanoliposomes, uniform and diffuse fluorescence in the cytoplasm of these cells, as well as in the nuclei, was observed.4. The concentration of cou-6 in the rabbit and mouse aqueous humor and corneas following topical administration of these two formulations showed: The cou-6 levels in corneas of the flexible liposomal formulation were all significantly higher than those of the liposomal formulation at different time point. From CLSM observation after multiple dosing, the corneal permeation of flexible nanoliposomes was stronger compared with nanoliposomes. Cou-6 in the aqueous humor was detected only in flexible nanoliposome group at some time points.5. No anti-inflammatory activity was detected either in the dipotassium glycyrrhizinate group(8.56mg/ml) or in the flexible nanoliposome group being modified by dipotassium glycyrrhizinate with the same concentration.Conclusions:The results of this investigation showed that the fluidity of flexible nanoliposomes containing dipotassium glycyrrhizinate was well and they should be safe for topical ocular application. their cellular uptake mechanism was changed after being modified, thus the capability for in vitro cell uptake and in vivo corneal penetration was significantly promoted. Therefore, flexible nanoliposomes might be a new promising drug delivery system for the treatment of ocular surface disorders. |
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