Chitosan Nanoparticles As A Potential Vehicle For Gatifloxacin In Ocular Delivery: Preparation, Characterization And Toxicity Concerns In Vivo

Objective To develop a convenient and reproducible method for preparation of gatifloxacin chitosan nanoparticles(GX-CSNPs) and to characterizatize in vitro and toxicity in vivo. To provide a new method for a new type ocular surface sustained release preparation. To provide correlated prophase experimentation accordings for ocular surface application of nanoparticles drugs.The study includes three parts:ExperimentⅠ:To prepare Gatifloxacin chitosan nanoparticles(GX-CSNPs) via ionotropic gelification..ExperimentⅡ: Study on the characterizatize in vitro about the GX-CSNPs.ExperimentⅢ: Study on toxicity in rabbit ocular surface about the GX-CSNPs.MethodsExperimentⅠ: It was optimised using design of experiments by employing a 3-factor, 3-level orthogonal experiment design. Independent variables were the amount of the bioadhesive polymers: chitosan (CS), tripolyphosphate(TPP) and the amount of gatifloxacin(GX) in the formulation. The dependent variables were the particle size, zetapotential, encapsulation efficiency and loading capacity.ExperimentⅡ: Morphology of the nanoparticles was detected by transmission electron microscope(TEM). The average size and zetapotential of the particles were detected by photon correlation spectroscopy. Moreover encapsulation efficiency and loading capacity were masured with the high performance liquid chromatography(HPLC). All data were evaluated using variance analysis to select the third group as the optimizing.ExperimentⅢ: To test toxicity in ocular surface, an in vivo local, acute tolerance assay was performed. Five female albino New Zealand rabbits weighing 2.0 to 2.5 kg were used. Animals received 30μL of the 3.0 mg/mL GX-CSNPs formulation in the right eye every 30 minutes for 6 hours. The contralateral left eye was used as the control and received physiological saline. 3, 6, and 24 hours after the first instillation, animal discomfort and clinical signs in conjunctiva, cornea, and lids were macroscopically evaluated by a trained ophthalmologist, according to a modification of the scoring system established in the Organization for Economic Cooperation and Development (OECD) guidelines(1987) for ocular irritation testing.The animals were euthanatized by deeply anesthetized by venous administration of an overdose of Sonistan (18 mg/kg), followed by air embolism. Cornea, conjunctiva and lid tissues were removed, fixed in formaldehyde and glutaric dialdehyde solution for a hematoxylin-eosin staining pathology study and transmission electron microscope respectively. Eyeball and lid sections were evaluated in a masked fashion according to the following criteria: alteration of the corneal, conjunctival epithelia, edema in lid tissues, presence of inflammatory eosinophils, neutrophils, mast cells or lymphocytes, and any other abnormality.ResultsExperimentⅠ: The GX-CSNPs are light blue opalescence suspension,inadequacy clear,neither production sediment,Tyndall experiment masculine.ExperimentⅡ: optimization group concentration as follow via orthogonal experiment:CS 0.10(% w/v),TPP 0.10(% w/v),GX0.15(% w/v)。The average particle size of nanoparticles was 232nm, zetapotential was 32mv, encapsulation efficiency was 67.04% and loading capacity was 11%. The morphology revealed regular well-identified spherical shape.ExperimentⅢ: Rabbits showed no signs of discomfort at 24 hours. Treated eyes had a clinical macroscopic sign score of 1, control eyes had a o. No differences were observed between treated and control eyes. Conclusion1.Ionotropic gelification is a convenient,good reproducibility method to prepare nanoparticles, and the Tyndall phenomenon can identity.2. We can get the optimal concentration matching via orthogonal experiment, and confirm the characters well via detection.3. GX-CSNPs are no acute toxicity to rabbit ocular surface,no Inflammatory reaction.