The Study Of Loaidng Tetrandrine And 1-Bromoheptadecaflurooctane Liposomes Treating With Dry Eye Disease

PART I PREPARATION AND CHARACTERIZATIONS OF LAODING TETRANDRINE AND 1-BROMOHEPTADECAFLUROOCTANE LIPOSOMESObjective To prepare a kind of loading tetrandrine and PFOB liposome(PFOB@LIP-Tet),with PFOB serving as the core and Tet inserted into the shell.To detect the primary physicochemical properties of PFOB@LIP-Tet and verify that Tet is successfully loaded on liposomes.Methods Using film dispersion-ultrasonic oscillation method to prepare PFOB@LIP-Tet,which loading Tet and shelling PFOB.Detect the primary physicochemical properties of PFOB@LIP-Tet.Detect the volume of Tet encapsuled in PFOB@LIP-Tet to calculate the encapsulation efficiency and loading capacity of Tet.Mean size with prolonged time duration for 7 days was detected to certify the stability of PFOB@LIP-Tet.Results PFOB@LIP and PFOB@LIP-Tet were successfully prepared and their mean size were respectively 79.4±7.7 nm and 103.4±7.8 nm.The measured zeta potential of PFOB@LIP and PFOB@LIP-Tet were respectively 6.0±1.2 m V and-21.8±0.6 m V.UV–vis–NIR spectrum showed that the absorbance of PFOB@LIP-Tet mainly come from Tet.Under TEM,we could see that liposome served as shell and PFOB was the core,with Tet inserted into the shell in PFOB@LIP-Tet.The differences between PFOB@LIP and PFOB@LIP-Tet in diameter,zeta potential and morphology under TEM showed that Tet was successfully loaded on the liposomes.The diameters of PFOB@LIP-Tet measured in succession for 7day were 87.5±9.15 nm,86.1±6.8 nm,103.4±7.8 nm,100.1±7.8nm,100.4±12.3 nm,83.4±4.3 nm and 97.1±0.6 nm,which showed that PFOB@LIP-Tet could be stored at 4℃ stably.Conclusions PFOB@LIP-Tet was successfully synthesized and its primary physicochemical properties were detected and stability at 4℃ was verified.PART Ⅱ DETECTION OF DRUG REEASE PATTERNS,BIOSAFETY,DISPERSION AND CALCULATION OF LOADING TETRANDRINE AND 1-BROMOHEPTADECAFLUROOCTANE LIPOSOMESObjective To detect the drug release curve of PFOB@LIP-Tet to certify the establish of controlled-system.To verify the safe concentrations of and Tet PFOB@LIP-Tet and treating concentration.To detect the dispersion and calculation of PFOB@LIP-Tet in vitro and in vivo.Methods HPLC was employed to detect the release volume of Tet at different time points.CCk-8 was employed to find out the safe concentration of Tet and PFOB@LIP-Tet,and then live/dead staining was used to verify this concentration.Then the calculation of PFOB@LIP-Tet in vitro was explored by detecting the fluorescence-labelled PFOB@LIP-Tet with CLSM,and the results was also analyzed quantitively by FCM.Then FLI system was used to compare the calculation of PFOB@LIP-Tet between the normal eye and DED eye indirectly and also the volume of PFOB@LIP-Tet dispersing on conjunctiva,cornea,iris,aqueous humor and crystalline lens of normal and DED eye.Results 75% of Tet was discharged from PFOB@LIP-Tet at 480 min.In vitro,the survival rate of rabbit corneal epithelial cells after incubated with Tet for 24 h or 48 h was beyond 50%,no matter free in PBS or encapsuled in PFOB@LIP-Tet.When incubation time was same,fluorescence signal intensity in normal rabbit corneal epithelial cells was far less than that in inflamed rabbit corneal epithelial cells.Fluorescence signal could hardly be detected in normal corneal epithelium,on contrast,strong fluorescence signal was detected in corneal epithelium of DED model.Two hours after administration,fluorescence signal was evident on conjunctiva and iris of DED eye,and only a little fluorescence signal was observed on the conjunctiva of normal eye.Conclusions Controlled-release system was successfully established.In vitro,when the concentration was 5 μg/ml,Tet was biosafe,regardless free or encapsuled in PFOB@LIP-Tet,in addition,biosafety of Tet was increased after loaded on PFOB@LIP-Tet.PFOB@LIP-Tet could be easily phagocytosed by inflamed rabbit corneal epithelial cells,and normal rabbit corneal epithelial cells hardly did.The accumulation of PFOB@LIP-Tet in DED eye was higher than that in normal eye,and most of PFOB@LIP-Tet dispersed in cornea and conjunctiva.PART Ⅲ STUDY OF THERAPEUTIC EFFECTS AND MECHNISM OF LOADING TETRANDRINE AND 1-BROMOHEPTADECAFLUROOCTANE LIPOSOMES IN TREAMENT OF DRY EYE DISEASEObjective To verify the anti-inflammation function of loading Tetrandrine and 1-Bromoheptadecaflurooctane liposomes formulations and their therapeutic effects on dry eye disease and impacts on intraocular pressure.Methods Establish inflammatory model of rabbit corneal epithelial cells,and divide them into four groups randomly,including control group,PFOB@LIP group,Tet group and PFOB@LIP-Tet group,which were respectively treated by PBS,PFOB@LIP,Tet and PFOB@LIP-Tet.The gene transcription of VEGF,IL-1β,TNF-α and PEG2.ELISA was employed to detect the concentration of VEGF,IL-1β,TNF-α and PEG2 released into culture mediums.Then the survival rates of inflamed rabbit corneal epithelial cells were analyzed.Dry eye disease models were established,28 totally,and divided into four groups,including ATS group,PFOB@LIP-ATS group,Tet-ATS group and PFOB@LIP-Tet-ATS group,respectively intervened by ATS,PFOB@LIP,Tet-ATS and PFOB@LIP-Tet-ATS,and then therapeutic effects of four groups were compared: cornea dyeing was observed under slit lamp,PAS staining of goblet cells was employed to observe the goblet cells in bulbar conjunctiva,H&E staining was used to compare the corneal epithelial thickness,Tunnel staining was employed to survey the apoptotic cells in corneal epithelium.Results In vitro,the expression level and concentration of VEGF,IL-1β,TNF-α and PEG2 in PFOB@LIP-Tet group was lowest,and the survival rates of PFOB@LIP-Tet group was highest.In vivo,corneal dyeing area and extent in PFOB@LIP-Tet-ATS group was smallest after intervened for same time after stained by sodium fluorescein.HE results showed corneal epithelium of PFOB@LIP-Tet-ATS group was thickest,while PAS staining showed that the number of goblet cells of the DED eye from PFOB@LIP-Tet-ATS group was largest and Tunnel staining results showed that apoptotic cells in corneal epithelium of PFOB@LIP-Tet-ATS group was fewest.Intraocular pressure changes of the DED eye in PFOB@LIP-Tet-ATS group and ATS group showed no statistical change before and after treatment.Conclusion PFOB@LIP-Tet showed significant therapeutic function on DED,and little impacts on intraocular pressure.