Alginate-Natamycin Slow-Release Ophthalmic Hydrogel For The Treatment Of Aspergillus Fumigatus Keratitis

Objective:To study the therapeutic effect of alginate-natamycin slow-release ophthalmic hydrogel to keratitis infected with Aspergillus fumigatus.Methods:1.natamycin-loaded hydrogel（SA-PEO-NATA hydrogel）was prepared using sodium alginate（SA）and polyethylene oxide（PEO）and cross-linked with four different ratios of calcium ion solutions（2:1,1:1,1:2,and 0:1 of ethanol to water,respectively）.2.The mechanical strength and ductility of the hydrogels were tested using a tensile test.The physical and chemical properties of the hydrogels were measured using fourier transform infrared（FTIR）and scanning electron microscope（SEM）.3.Ultraviolet-visible spectrophotometry was used to compare the in vitro NATA release capacity of the four hydrogels.4.The effect of hydrogel on the growth and survival of human corneal epithelial cells was assessed by CCK-8 method.Corneal irritation of C57BL/6 mice was evaluated by Draize ophthalmic toxicity test.5.The prepared hydrogel was placed at 4℃and room temperature for one month,and the inhibitory effect of hydrogel on the growth of Aspergillus fumigatus in vitro was measured by inhibition zone method.6.The corneas of C57BL/6 mice infected with Aspergillus fumigatus were treated with SA-PEO-NATA hydrogels to assess the clinical scores after infection.The fungal load in the corneas of infected mice was detected by colony count method,and the infiltration of inflammatory cells in the corneas of infected mice was observed by hematoxylin-eosin staining.7.RT-PCR and ELISA tests were used to detect the mRNA and protein expression of IL-6,TNF-αand MIP-2 in RAW 264.7 cells.Results:1.At the speed of 10mm/min,the mechanical strength of SA-PEO-NATA hydrogel decreased gradually with the decrease of ethanol content in crosslinking solution.The 2:1 ethanol/water hydrogel could withstand the maximum mechanical strength,which could withstand the maximum tensile force of 6.392N,the strength of0.66MPa,the elastic modulus of 0.57MPa,and the ductility of 100%.FTIR analysis showed the successful cross-linking of hydrogels with calcium ions and the presence of natamycin in SA-PEO-NATA hydrogels by physical adsorption.SEM showed that the surface of hydrogel crosslinked with Ca²⁺solution of 2:1 ethanol/water was smoother and denser compared with hydrogel crosslinked with Ca²⁺solution of 0:1 ethanol/water and the evidence of natamycin on the surface of the hydrogel.2.CCK-8 results indicated that the four kinds of hydrogels exerted no inhibitory effect on cell growth.The results of Draize ophthalmic toxicity test demonstrated that hydrogel did not stimulate the cornea of mice.4.The results of the in vitro release assay showed that the four hydrogels released up to 96%of natamycin in 24 hours,with the slowest release rate being the ethanol/water 2:1 hydrogel and the ethanol/water 0:1 hydrogel being the fastest release rate group.5.The inhibition zone of four SA-PEO-NATA hydrogels was significantly larger than that of the same dose of natamycin group.The blank control group did not show any inhibitory zone.The inhibition zone formed by four kinds of SA-PEO-NATA hydrogels was still larger than that in the same dose of NATA group 6.Compared with the infected group,SA-PEO-NATA hydrogel treatment for 5 days resulted in lower corneal inflammation scores,reduced area of cloudiness,reduced infiltration of inflammatory cells and reduced number of fungal colonies in the mouse corneas.7.SA-PEO hydrogel significantly reduced cellular IL-6,TNF-αand MIP-2 mRNA and protein expression after Aspergillus fumigatus stimulated RAW 264.7 cells.Conclusion:SA-PEO-NATA ophthalmic hydrogel has remarkable biocompatibility,antibacterial and anti-inflammatory effects,which can effectively treat mice Aspergillus fumigatus keratitis.Hydrogels with 2:1 ethanol to water ratio optimally sustained the release of NATA.In conclusion,SA-PEO-NATA ophthalmic hydrogel with 2:1ethanol/water is a feasible treatment for fungal keratitis.