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. Author manuscript; available in PMC: 2024 May 1.
Published in final edited form as: Adv Drug Deliv Rev. 2023 Mar 17;196:114776. doi: 10.1016/j.addr.2023.114776

Fig. 2. Construction of a Dectin-1-CRD coated Amphotericin B loaded pegylated liposome, DEC1-AmB-LL.

Fig. 2.

DEC1-AmB-LL serves as an example DectiSome. (A). Steps involved in Dectin-1 cloning, expression, purification, modification, and insertion into the liposome membrane are shown. 1. Synthetic DNA encoding a truncated fragment of Dectin-1 (DEC1) including the CRD and stalk region and N-terminal his6 and lysine (Lys) tags, but lacking the membrane spanning and signaling domain, was cloned into an E. coli plasmid expression vector. 2. Dectin proteins from expressing E. coli cells were extracted into 6 M guanidine hydrochloride (GuHCl) with beta-mercaptoethanol (BME) generating random coiled protein with its six reduced cysteine residues. The random coiled protein was affinity purified on Ni(II) resin. 3. A pegylated lipid moiety, PEG-DSPE, was coupled to the Lys tag in the denatured proteins. Coupling reagents and GuHCl were removed by gel exclusion chromatography into a 1 M Arginine BME crowding buffer that is proposed to partially renature the protein and improve its final renaturation in biological buffers. 4. DEC1-AmB-LLs were assembled by incubating a pegylated analog of AmBisome®, AmB-LLs, the lipid modified DEC1 protein, and lipid modified rhodamine at 60°C, the transition temperature of these liposomes. When these DectiSomes are diluted into biological buffers, the liposomal DEC1 is proposed to reform three cystine bridges necessary for proper protein folding. (B). A DEC1-AmB-LL is shown binding its cognate ligand an oligoglucan (green). The typical structure and composition of a DectiSome are indicated. Based on the initial lipid moieties of an un-pegylated liposome representing 100 moles percent of lipid, the proportion of each additional reagent is shown and its mole percent indicated, diluting the unmodified liposomal lipids to 83.5 moles percent. Each 100 nm diameter liposome contains approximately 1,500 molecules of Dectin-1, 3,000 of Rhodamine-DHPE, 3,000 of PEG-DHPE, and 17,000 of AmB. The DEC1-PEG-DSPE monomers are free to float in the liposome membrane and form active dimers that bind Dectin-1’s cognate oligoglucans [107]. Alternate functional AmB-loaded DectiSomes coated with the CRDs of Dectin-2, Dectin-3 and DC-SIGN have been constructed similarly [215,217,220]. Dectin-2 coated liposomes containing Anidulafungin instead of AmB have also been assembled by related methods [218].