DC-SIGN targets amphotericin B-loaded liposomes to diverse pathogenic fungi

Abstract

Background Life-threatening invasive fungal infections are treated with antifungal drugs such as Amphotericin B (AmB) loaded liposomes. Our goal herein was to show that targeting liposomal AmB to fungal cells with the C-type lectin pathogen recognition receptor DC-SIGN improves antifungal activity. DC-SIGN binds variously crosslinked mannose-rich and fucosylated glycans and lipomannans that are expressed by helminth, protist, fungal, bacterial and viral pathogens including three of the most life-threatening fungi, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Ligand recognition by human DC-SIGN is provided by a carbohydrate recognition domain (CRD) linked to the membrane transit and signaling sequences. Different combinations of the eight neck repeats (NR1 to NR8) expressed in different protein isoforms may alter the orientation of the CRD to enhance its binding to different glycans. Results We prepared two recombinant isoforms combining the CRD with NR1 and NR2 in isoform DCS12 and with NR7 and NR8 in isoform DCS78 and coupled them to a lipid carrier. These constructs were inserted into the membrane of pegylated AmB loaded liposomes AmB-LLs to produce DCS12-AmB-LLs and DCS78-AmB-LLs. Relative to AmB-LLs and Bovine Serum Albumin coated BSA-AmB-LLs, DCS12-AmB-LLs and DCS78-AmB-LLs bound more efficiently to the exopolysaccharide matrices produced by A. fumigatus, C. albicans and C. neoformans in vitro, with DCS12-AmB-LLs performing better than DCS78-AmB-LLs. DCS12-AmB-LLs inhibited and/or killed all three species in vitro significantly better than AmB-LLs or BSA-AmB-LLs. In mouse models of invasive candidiasis and pulmonary aspergillosis, one low dose of DCS12-AmB-LLs significantly reduced the fungal burden in the kidneys and lungs, respectively, several-fold relative to AmB-LLs. Conclusions DC-SIGN’s CRD specifically targeted antifungal liposomes to three highly evolutionarily diverse pathogenic fungi and enhanced the antifungal efficacy of liposomal AmB both in vitro and in vivo. Targeting significantly reduced the effective dose of antifungal drug, which may reduce drug toxicity, be effective in overcoming dose dependent drug resistance, and more effectively kill persister cells. In addition to fungi, DC-SIGN targeting of liposomal packaged anti-infectives have the potential to alter treatment paradigms for a wide variety of pathogens from different kingdoms including protozoans, helminths, bacteria, and viruses which express its cognate ligands.

Dectin‐3‐targeted antifungal liposomes efficiently bind and kill diverse fungal pathogens

“…The E. coli ‐produced DEC3 polypeptide was affinity purified (Figure SF2a,b ) and coupled to a pegylated lipid to make DEC3‐PEG‐DSPE. The DEC3‐PEG‐DSPE polypeptide was inserted via its DSPE lipid moiety into the AmB‐LLs at 1 mole percent to make the DectiSome DEC3‐AmB‐LL by methods we have described previously for other DectiSomes (Ambati, Ellis, et al, 2019 ; Ambati, Ferarro, et al, 2019 ; Ambati, Pham, et al, 2021 ). The construct allows the CRD and stalk region of DEC3 to float freely in the liposomal membrane and to form functional homodimers and multimers (Meagher et al, 2021 , 2023 ).…”

“…The areas of rhodamine B red fluorescence were measured from multiple photographs to quantify these binding data. We began assessing fluorescent liposome binding area data by manually processing each image through ImageJ as done previously (Ambati, Ellis, et al, 2019 ; Ambati, Ferarro, et al, 2019 ; Ambati, Pham, et al, 2021 ; Choudhury et al, 2022 ). To reduce the labor involved in quantifying fluorescent liposome binding data and standardize the process, we created a CellProfiler pipeline, AreaPipe, that automates quantitative image analysis (Figure SF3 ).…”

“…DectiSomes have several distinct advantages over more classical antibody‐targeted immunoliposomes, including a wider range of cognate ligands, lower cost, and greater avidity (Meagher et al, 2021 , 2023 ). We have previously demonstrated the potential of three CTLs, Dectin‐1 ( CLEC7A ), Dectin‐2 ( CLEC6A ), and DC‐SIGN ( CD209 ), to efficiently target Amphotericin B‐loaded DectiSomes to four human fungal pathogens: C. albicans, R. delemar, C. neoformans , and Aspergillus fumigatus (Ambati et al, 2022 ; Ambati, Ellis, et al, 2019 , 2021 ; Ambati, Ferarro, et al, 2019 ; Ambati, Pham, et al, 2021 ; Choudhury et al, 2022 ; Meagher et al, 2021 ). One or more of the CTL‐targeted DectiSomes dramatically lowered the effective dose of Amphotericin B (AmB) when these pathogens were grown in vitro.…”

“…One or more of the CTL‐targeted DectiSomes dramatically lowered the effective dose of Amphotericin B (AmB) when these pathogens were grown in vitro. DectiSomes were also shown to be effective at reducing fungal burden and/or improving mouse survival in murine models of aspergillosis and candidiasis (Ambati et al, 2022 ; Ambati, Ellis, et al, 2021 ; Ambati, Pham, et al, 2021 ).…”

Nanoliposomes as Drug Delivery Systems for Antifungal Therapy

“…Furthermore, DCS12-AmB-LLs inhibited and/or killed all three species more effectively than AmB-LLs or BSA-AmB-LLs. In mice models of invasive candidiasis and pulmonary aspergillosis, a single low dose of DCS12-AmB-LLs decreased the fungal burden in the kidneys and lungs several-fold more than AmB-LLs (Figure 2) [57] Santoso et al ( 2022) have recently developed AmB-loaded hybrid NLPs decorated with engineered chitin-binding domain (LysM) of P. ryukyuensis chitinase. Because of its specificity toward chitin, which is absent in humans but abundant in fungal cell walls, the enzyme chitinase (EC.…”