| ObjectiveTo develop a new Drug Delivery System—Amphotericin B liposome-fibrin glue-amniotic membrane (AFAM). Observe its morphology, study the slow-release and fungistasis of which in vivo and vitro. Provide experimental foundation for development and clinical application of drug delivery in the future.Experiment I: study on the preparation and morphology of the AFAM.Experiment II: study on the slow release of AFAM in vivo by high performance liquid chromatography (HPLC).Experiment III: study on the antifungal activities of AFAM in vitro.Experiment IV: study on the aqueous humor drug concentration after AFAM transplantation.MethodsExperiment I: We added 1000 mg Amphotericin B liposome (containing 10 mg Amphotericin B ) into 2.5 ml fibrin glue (FG) catalyst, and admixed with 2.5 ml FG host, then applied appropriate amount of adhesive solution to the entire basal surface of amniotic membrane (AM) and freeze drying, to prepare the AFAM, of which concentration is 2 mg/ml and thickness is 0.2mm～0.3 mm. Observe morphology of AFAM with Light Microscope and Scanning Electron Microscope (SEM).Experiment II: AFAM was stored in the saline (5 ml) at 37℃. Saline was replaced every 24 hours,and till the 10 day. Preserved saline containing the released antifungal was stored at - 40℃until essay by high performance liquid chromatography (HPLC).Experiment III: Aspergillus fumigatus strains were inoculated onto potato dextrose agar (PDA) at 25～28℃, and Fusarium solani strains were inoculated onto Czapek's agar (CA) at 25～28℃, then prepared the spore suspension. According to broth microdilution antifungal susceptibility test, we got sterile 96 holes culture plate, the 1-11 holes in 1-4 rows were added into 100μl antifungal physic liquor with RPMI-1640 broth multiple proportion diluted, the 1-10 holes in 5-8 rows were added into 100μl replaced saline of each day, the 12 holes of each row add blank saline as control, then added 100μl prepared spore suspension into each hole of the culture plate. The culture plate were incubated 46～50 hours at 35℃, and interpreted the MIC result.Experiment IV: 15 New Zealand rabbits were divided into 3 groups. The AFAMs were transplanted to the ocular surface of right eyes, topical 2 mg/ml AmBL solution was applied to the left eyes as control, 3 times daily. Collected 100μl aqueous humor at the same time on 1,2,3,4,5 day after transplant and eyedrops, 1 rabbit 6 eyes were obtained each group and each time. The aqueous humor AmB concentration was assayed by HPLC.ResultsExperiment I: Appearance: The AFAM was yellow, very thin and smooth like a paper, it could retain tenacity and flexible on hydration. HE stain: AmBL-FG was tight combined with the chorionic surface of amniotic membrane, simple columnar epithelium cells were to line up in order on epithelium surface of AM, AmBL-FG was stained uniformly, the thickness of which was 0.3 mm. The FG collagen fiber was to be visible network structure. SEM: the homogeneous, reticular network of fibrin glue was readily visible and revealed irregularly sized pores ranging from 6～24μm in diameter. AmBL crystals were to be trapped by the FG matrix as solid particles extending from within the fibrin glue.Experiment II: The FG in AFAM was degradation completely at 10 day. AmB was sustained release from AFAM with FG slow degradation, AmB concentration in saline at first 3 days were 59.9μg/ml,34.1μg/ml,27.9μg/ml, release rate accumulate to 30.38 %, then gradually reduced. AmB concentration in saline was 0.78μg/ml at the 10 day, which was in concentrations of minimum inhibitory concentration (MIC) to most fungus.Experiment III: The minimum inhibitory concentrations (MICs) of Amphotericin B liposome (AmBL) against Fusarium solani were range from 1.0 to 2.0μg/ml, against Aspergillus fumigatus were range from 0.5 to 2.0μg/ml. The MICs of saline containing AmB against Fusarium solani were 1.5 to 2.0μg/ml, against Aspergillus fumigatus were 0.78 to 2.0μg/ml.Experiment IV: After transplantation, AFAM became thinning along with FG slow degradation, the FG was degraded completely till 5 day. The aqueous humor AmB concentration at first 2 days were 113.1 ng/ml,61.2 ng/ml, then gradually reduced, which could still maintained at 28.0 ng/ml at 5 day. In control group, aqueous humor AmB concentration was 26.1ng/ml on average and kept a low but stable level.Conclusions1. The AFAM was to be trapped a large amount of drug, which possess stable biology and physics performance.2. With the degration of FG, AmB could be released gradually, whose effect of sustained release in vitro is detected by HPLC.3. The antifungal activities in vitro revealed that AFAM could fungistasis persistently, and fungistatic activity got weaker gradually.4. After AFAM transplantation, the concentration of AmB could be detected in aqueous humor, which demonstrated that the drug in the AFAM could be released gradually and penetrated into eye.
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