The Resistant Effect Of Chitosan-zymosan Microspheres On Candida Albicans In Vivo

ObjectiveTo observe the effects of chitosan and zymosan on C. albicans.Microspheres were prepared with chitosan and zymosan, and investigatetheir effects in rats with C. albicans pneumonia.Methods1. XTT reduction assay was used to evaluate chitosan susceptibility ofC. albicans biofilm growth activity in96-well plates. Fluorescencemicroscopy and scanning electron microscopy were used to observe theinfluence of chitosan on dead/vital cells and morphology of biofilm. TotalRNA was extracted from lung tissue of rats which were injected withdifferent concentrations of zymosan and was measured the expression ofDectin-1with PCR.2. Chitosan-zymosan microsphere was prepared by cross-linkingemulsification method. Through analyzed some factors of preparationprocess: drug ratio, oil-water ratio, the stirring speed and crosslinking agent,the optimum preparation conditions were selected. The character andsurface of microspheres were investigated by SEM and Fourier TransformInfrared Spectroscopy (FTIR) 3. The model of pulmonary infection by C. albicans was established inimmunocompromised rats, the rats were randomly divided into four groups:model group, chitosan group, combination group and chitosan-zymosanmicrosphere group. According therapy was given to random-divided groups,the histopathologic changes in lung tissue of each group were observedusing HE stain. The levels of Dectin-1and TLR2mRNA were examined byreal-time PCR and the levels of IL-10and TNF-α were examined byELISA.Results1. The effects of0.0313%chitosan on inhibition against the growth ofC. albicans planktonic cells and early phase of biofilm were significant.With the maturing of the biofilm,0.0625%chitosan on biofilm was moresignificantly inhibitory effect. Fluorescence microscopy and scanningelectron microscopy showed that0.0625%chitosan has not only the abilityto damage the vast majority fungal cell growth, but also delay theformation of biofilms. Dectin-1expression in rats dramatically increasedafter2%,1%and0.5%zymosan induction, but high concentration ofzymosan might be activated systemic inflammatory response.2. The cross-linking emulsification method was stable and simpleoperation. The microspheres were prepared under the condition of thepreparation：the ratio of drug was3:2, oil-water ratio was8:1, the stirringspeed was600rpm and crosslinking agent was glutaraldehyde saturated toluene solution, which was shown to have good sphere, good physicaldispersivity, basic uniform size by SEM. FTIR data also supported thatChitosan-zymosan microsphere was successfully formed by zymosan andcross-linking of chitosan.3. Chitosan-zymosan microsphere was used in treatment of C. albicanspneumoniae. The inflammatory changes in the lung tissues were obviouslylightened in rats treated with chitosan-zymosan microsphere. Comparedwith the chitosan group and the model group, the chitosan-zymosanmicrosphere group had significantly increased Dectin-1, TLR2and IL-10levels, but level of TNF-α had dramatically decreases.Conclusion1. Chitosan can effectively kill C. albicans and inhibit its biofilmformation in vitro. Zymosan can induce Dectin-1expression up-regulation2. The preparation process of microspheres by cross-linkingemulsification method is simple. The optimized technique is suitable forthe preparation of microspheres3. Chitosan-zymosan microsphere can increase expression of TLR2and Dectin-1in the rats infected with C. albicans pneumonia, promote thesecretion of IL-10and adjust the inhibition of TNF-α. It suggested thatthese mechanisms were involved in the therapeutical effects ofchitosan-zymosan microsphere on C. albicans pneumonia of theimmunocompromised rats.