Mannosylated Nanomicelles Bearing Amphotericin B And Doxorubicin For Enhanced Visceral Leishmaniasis Therapy

Leishmaniasis is an infectious disease caused by Leishmania spp.parasites.So far,there is no vaccine available,and clinical treatments are limited for their severe side effects,long treatment terms,drug resistance,and patient’s coinfection with HIV.To overcome these problems and improve the therapeutic efficacy,we designed and synthesized an amphiphilic molecule Man7-β-CD-C₃（MCC）and fabricated a nanosized drug-loaded micelle AmB&DOX@MCC by co-dialysis with the hydrophobic drugs amphotericin B（AmB）and doxorubicin（DOX）,in which the drug loading capacity and encapsulation efficiency were 9.8%&57.9%for AmB and 6.9%&41.1%for DOX,separately.The AmB&DOX@MCC micelles were spherical and uniformly dispersed with a diameter of about 30 nm under a transmission electron microscope,had the hydrodynamic size at about 167nm in Dulbecco’s phosphate-buffered saline,held a slipping plane potential at about-31.6 mV in ultrapure water,and remained stable within 7 days in complete medium and DPBS.Meanwhile,the AmB&DOX@MCC micelles showed an acid-stimulated drug release profile,which can release drugs quickly and completely（>50%in 24 hours and>70%in 72 hours）in DPBS with pH=4.5.The AmB&DOX@MCC micelles also had good biosafety.In the MTT assay,the cell viability of RAW 264.7 cells and HEK 293 cells were higher than 80%after incubated with it for48 hours.In the hemolysis assay,the hemolytic rate of red blood cells was less than 1%after incubated it for 2 hours.At the same time,the AmB&DOX@MCC micelles had strong actively targeted drug delivery capabilities.In the qualitative and quantitative experiments of confocal laser scanning microscopy and flow cytometry,RAW 264.7 cells（mannose receptors high expression）showed a significantly higher ability to uptake AmB&DOX@MCC micelles than RAW 264.7 cells pretreated with mannose solution or HEK 293 cells（mannose receptors low expression）.Finally,AmB&DOX@MCC micelles had excellent anti-leishmaniasis efficacy.In the cell infection model of Leishmania donovani parasites,the drug efficacy(IC_50AmB:18.2±2.6 ng·mL^-1,IC_80AmB:25.1±3.6 ng·mL^-1,IC_50DOX:13.0±1.8 ng·mL^-1,IC_80DOX:17.9±2.6 ng·mL^-1)of AmB&DOX@MCC micelles were significantly better than non-targeting free and liposomal drug combinations or single drug-loaded nanomicelles.AmB&DOX@MCC micelles are promising in the future treatment of Leishmaniasis.