| Up to now, the therapeutic method of ocular diseases such as proliferative retinopathy, uveitis and cytomegalovirus retinitis is still a major clinical problem. Doxorubicin as an anti-proliferative drug has been used in proliferative retinopathy in some experiments. Because of the particularity of local application, drugs need to provide a sufficient stability. The widely used eye drops, which have short duration and low concentration of the therapeutic action of topically administered drugs, have seriously restricted the therapeutic effect. For this reason, the research on new ophthalmic preparations which can enhance the bioavailability and duration becomes more and more important. It has been demonstrated that thiolated chitosan (TCS), a derivative of chitosan (CS), is a new kind of ophthalmic delivery carrier due to its water solubility, mucoadhesive ability and gelling properties in situ at physiological pH. Besides, a natural proteoglycan hyaluronan (HA), discovered in extracellular matrix and connective tissue, has been considered as a biocompatible material. In this study, we prepared TCS-HA nanoparticles loaded doxorubicin as a new ocular drug delivery system.Six parts included in this thesis:1. The establishment of Doxorubicin (DOX) analysis using fluorometric assayWe had invesgated the specificity, accuracy, linear equation and the average recovery of DOX using fluorometric assay, establishing a high sensitive and accurate method to measure DOX concentration.2. The synthesis and evaluation of TCS and the degradation of HAWe used the previous method discovered by our lab to synthesize TCS. The degree of thiol immobilized was838.71±5.13μmol/g measured by Ellman’s reagent. The HA had been degraded from150wDa to50wDa using hydrochloric acid according previous reports. The molecular weight of degraded HA was homogeneous distribution measured by HPGPC.3. Preparation and characterization of DOX-NPsThe study prepared the nanoparticles by a ionic gelation method which was sustained and controlled released. The size of DOX-NPs was257nm, and the stability assay of this nanoparticles turned out well. 4. In vitro cytotoxity study and cell uptake assay of human corneal epithelium cells (HCE)The cytotoxicity of DOX-NPs showed much safer than DOX solution measured by MTT. The DOX-NPs exhibited highly uptaken by HCE compared to DOX water solution photoed by laser scanning confocal microscope. And it is time-dependent.5. In vivo studiesComparing corneal and sclera permeation ability of DOX solution and DOX-loaded TCS-HA NPs in vivo, we further confirmed that NPs can enhance the drug permeation, indicated that TCS-HA NPs would be an effective potential system for ocular drug delivery.Conclusion:We prepared TCS-HA NPs loaded DOX, which carried out with stability, controlled released property, low cytotoxity and high permeation in corneal and sclera. The research may provided groundwork for a strategy developping new material and ophthalmic mucoadhesive preparation. |
