Study Of The Preparation Of Diosmetin-Loaded Micellespolymer Micelles And Its Anti-Cataract Effect

In this paper,PAMAM-PEG₁₀₀₀-DSPE（PPD）was prepared from NHS-PEG₁₀₀₀-DSPE and PAMAM by hydrolysis.The concentration of PAMAM in PPD was obtained quantitatively by the reaction of amino group on PAMAM and2,4,6-trinitrobenzene acid（TNBS）.The concentration of PAMAM in PPD was 0.259mmol/L.Three cationic polymers PPD,N,N,N-trimethyl chitosan（TMC）and（2,3-dioleoxy-propyl）trimethyl ammonium chloride（DOTAP）were combined with amphiphilic phospholipid vitamin E polyethylene glycol succinate（TPGS）and distearyl phosphatidyl ethanolamine giycol 2000(DSPE-PEG₂₀₀₀),respectively.Diosmetin was used as the model drug.Micelles with DOTAP or PPD as cationic materials（D-d-M,D-p-M）and TMC coated cationic micelles（D-m-T）were prepared by thin film hydration method and steric absorption.It was observed by transmission electron microscope that the micelles of D-d-M,D-m-T and D-p-M were regular in morphology and uniform in size distribution.The particle sizes of D-d-M,D-m-T and D-p-M micelles measured by Malvern instrument are（28.42±0.1131）,（29.28±0.48）,and（24.95±0.145）nm;the Zeta potentials were（6.01±1.14）,（6.46±0.0566）,and（6.19±0.117）m V,respectively.The encapsulation rates determined by centrifugation of D-d-M,D-m-T and D-p-M micelles were（97.46±0.35）%,（94.90±0.41）%and（94.78±0.43）%,respectively.The release of micelles in vitro was investigated by dialysis.The results showed that the Dio solution exhibited obvious sudden release,while D-d-M,D-m-T and D-p-M showed no sudden release.The cumulative release rate of Dio solution reached 84.77%at 4 h,while the cumulative release rate of D-p-M,D-d-M and D-m-T were only 26.59%,10.85%and 13.12%.After 12 hours,the cumulative release rate of Dio solution was more than 95%,while that of D-p-M,D-d-M and D-m-T was 64.35%,34.91%and 35.34%,respectively.After the Dio was encapsulated into micelles,its cell toxicity was lower than that of the free drug by CCK-8 method,and there was no obvious toxicity at the administration concentration.The irritation effects of D-d-M,D-m-T and D-p-M to eye were evaluated by Draize rabbit eye irritation experiment and H&E pathological section staining experiment.The results showed that the eye tissue structure was mantained,and the cells were arranged neatly and evenly,indicating that D-d-M,D-m-T and D-p-M had no visible irritation on rabbit eyes.Coumarin 6（Coumarin,Cou-6）was used as a fluorescence probe instead of Dio to prepare C-d-M,C-p-M and C-m-T micelles,and cellular uptake experiments were carried out.The fluorescence intensity of C-d-M,C-p-M and C-m-T in cells was 2.14,2.93,4.10 times as that of Cou-6 solution.Among them,the cellular uptake of C-m-T is the strongest.The precorneal retention of each preparation was observed by IVIS spectrum.It was found that the fluorescence signal of C-d-M,C-m-T and C-p-M labeled by Cou-6 within 20 min was eliminated by 36.05%,18.80%and 27.74%,respectively,while the fluorescence of Cou-6 solution eliminated by 53.19%.The corneal permeability of each preparation was examined by corneal penetration cells.The results showed that it was difficult for Dio solution to penetrate the cornea,and the corneal permeability content and steady flow rate of D-m-T were significantly higher than those of D-d-M and D-p-M,and the apparent permeability coefficient was 3.11 and 1.49 times as high as that of D-d-M and D-p-M,respectively.Corneal penetration experiments were conducted on C-d-M,C-p-M and C-m-T prepared by using Cou-6 for Dio,and the corneal penetration depth of fluoresceous-labeled preparations were compared.It was observed under confocal laser scanning microscope that the fluorescence penetration depth of the preparation increased with the time.At 2 h and 4 h,the fluorescence penetration of the three groups was deeper than that of Cou-6 solution group,and the penetration depth of C-m-T group in corneas was deeper than that of C-d-M and C-p-M group.The results of horizontal scanning of cornea at different depths showed that the fluorescence signals were mainly distributed around the cells,and a few were inside the cells,indicating that nano-preparation can pennetrate through the cornea by the paracellular bypass.To investigate the anti-cataract effect of D-d-M,D-m-T and D-p-M micelles,the lens opacity,catalase（CAT）,malondialdehyde（MDA）and superoxide dismutase（SOD）activities were determined.The results showed that D-d-M,D-m-T and D-p-M could delay the development of lens opacity to some extent,and the effect of D-m-T was better than that of D-d-M and D-p-M.The results of CAT activity test showed that compared with the blank control group,the CAT activity of the model group decreased significantly,and there was significant difference.Compared with the model group,CAT activity in the lens of rats in D-d-M,D-m-T and D-p-M treatment groups was significantly increased by 36.89%,45.50%and 45.99%,respectively.The results of MDA content determination showed that compared with the blank control group,the MDA content in the model group increased significantly.Compared with the model group,the MDA in D-d-M,D-p-M and D-m-T groups decreased by 44.60%,50.90%and 66.12%,respectively.The SOD content determination results showed that the SOD activity of the model group was significantly lower than that of the blank control group.Compared with the model group,SOD activities in D-d-M,D-m-T and D-p-M groups were increased,and SOD activities in D-m-T group were 1.11 times and 1.04 times of those in D-d-M and D-p-M groups,respectively,indicating that micelles coated with TMC could more effectively inhibit the oxidative damage caused by free radicals.