Background Voriconazole is the traditionally used antifungal agent,but its ophthalmic form is unsatisfactory.A novel ophthalmic drug delivery system with biomedical devices may be of promising for the prognosis of fungal keratitis.Objective This study was to investigate the sustained release,therapeutic effect and biocompatibility of effect and quaternized chitosan functionalized with carboxylated graphene and nano-silver and voriconazole (CS-ETA/Ag/GO/Vor) for fungal keratitis.Methods This study complied with the Regulations for the Administration of Affair Concerning Experimental Animals of State Science and Technology Commission.Two hundred and ten SPF female C57BL/6 mice were selected with the age 6-8 weeks for the biocompatibility experiment (30 mice) and therapeutic observation of CS-ETA/Ag/GO/Vor (180 mice).CS-ETA/Ag/GO and CS-ETA/Ag/GO/Vor were attached on the normal corneas of mice and compared with the normal mice to assess the histopathological changes.Aspergillus fumigatus-infected mouse models were established in the left eyes of 180 mice by intrastromally injection of 2.0 μl Aspergillus fumigatus suspension with the density of 5 × 107 CFU/ml,then the mice were randomized into the model control group,CS-ETA/Ag/GO group and CS-ETA/Ag/GO/Vor group,and the corresponding membrane were attached the central corneas in different groups.In 1 day,3,5,7 days after modeling,the corneas were examined under the slit lamp microscope and scored,and corneal sections were prepared for the histopathological examination.Fungal activity was confirmed by plate counts,and real-time PCR was employed to assay the relative expressions of interleukin-1β (IL-1β) mRNA and tumor necrosis factor-α (TNF-α) mRNA in the corneas.Results No morphological abnormality was seen in the corneas in the normal control group,CS-ETA/Ag/GO group and CSETA/Ag/GO/Vor group.Corneal inflammatory score was significantly lower in the CS-ETA/Ag/GO/Vor group in various time points,with a significant differences among the groups and time points (Fgroup =237.29,P=0.00;Ftime =260.33,P=0.00).The edema,necrosis or perforation of cornea were seen in the model control group,and slighter inflammatory response in the CS-ETA/Ag/GO group,and corneal edema was gradually disappear in the CS-ETA/Ag/GO/Vor group.The corneal fungal loads were highest in the model control group and lowest in the CS-ETA/Ag/GO/V or group,with significant differences among the three groups and various time points (Fgroup =113.15,P =0.00;Ftime =126.52,P=0.00).The relative expressions of IL-1β mRNA and TNF-α mRNA in the corneas peaked in the fifth day after modeling in all of the three groups,and the expression levels of IL-1β mRNA and TNF-α mRNA in the corneas were lowest in the CS-ETA/Ag/GO/Vor group,showing significant differences among the groups and time points (IL-1β:Fgroup =189.90,P =0.00;Ftime =108.56,P =0.00;TNF-α:Fgroup =82.55,P =0.00;Ftime =44.36,P =0.00).Conclusions CS-ETA/Ag/GO/Vor delivery system plays an anti-fungal activity in fungal keratitis by the synergistic effect of voriconazole and Ag+.In addition,CS-ETA/Ag/GO/Vor appears to have a good safety after topical application.

Objective To develop nanometer-scale bubbles with surfaces of N-palmitoyl chitosan(PLCS) as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo. Methods The PLCS nanobubbles were prepared using a cutting technique at differential high-frequency of shear speed. Both optical and transmission electron micrography were performed to determine the nanobubble size and morphology. Concentration, size-distribution and zeta potential of the PLCS nanobubbles were measured by cell counting chamber, Malvern lazer particle analyzer and zeta-sizer at 1-day, 45-day and 90-day. The acoustic effects of the PLCS nanobubbles on myocardium and renal tissue in 6 normal rats were observed using bolus infusion of the nanobubbles intravenously. The maximum video intensity(VI) was measured.Results The PLCS nanobubbles with nice round-shape and uniform site-distribution were demonstrated.The mean diameter,concentration and zeta potential of the PLCS nanobubbles were (617 ± 12) nm, (7.2 ±0.6) × 109/ml and (52.9 ± 1.3)mV at the 1-day,and all of parameters did not change significantly in 45-day and 90-day ( P ＞ 0. 05). A significant contrast-enhancement was noted on myocardium and renal tissue during infusion of the nanobubbles. VI on both tissues was (15.6 ± 1.1)GU and (27.3 ± 2.5)GU,respectively. The visual contrast-enhancement last up to (10 ± 2)min. Conclusions The PLCS nanometerscale bubbles have excellent physical-features and contrast-enhanced ultrasound effects in vivo. It may develop as a novel contrast ultrasound agent which could cross endothelial cell membrances.