Protein−Liposome Conjugates Using Cysteine-Lipids And Native Chemical Ligation

Abstract

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.

Morphology, binding behavior and MR‐properties of paramagnetic collagen‐binding liposomes

“…Using a protein determination the amount of coupled protein was determined to be 1.58 nmol of CNA35 per 1 mmol lipid. Assuming a surface area of 0.6 nm 2 per lipid and unilaminar liposomes with a diameter of 200 nm (circa 420,000 lipids per liposome), approximately 660 copies of CNA35 were estimated to be present per liposome (33). A ratio of 1 CNA35 to 15 Mal-PEG2000-DSPE molecules was used, which would correspond to 700 proteins per liposome, therefore the coupling efficiency was around 95%.…”

Surface Engineering Using Peptide Amphiphiles

“…104,105 The native chemical ligation (NCL) method 106,107 was employed successfully to create covalent linkages between recombinant proteins and the PEG-conjugated vesicles and micelles; NCL exclusively occurs at the protein C-terminus -often not involved in any binding activities with high specificity. These examples ( Figure 12) demonstrate a highly effective instillation of multiple functionalities (i.e., multiple collagen binding sites) into soft nanoparticles that may be necessary for targeting sites with multivalent architectures.…”

Biopolymer, Dendrimer, and Liposome Nanoplatforms for Optical Molecular Imaging

“…Recently, it was also reported that liposomes consisting of hepatitis B surface antigen and fluorescently labeled polystyrene beads or plasmids were used for tissue and cellular targeting with high specificity [88]. Another method of liposome ligation was reported by Reulen et al [89], which showed fluorescent proteins covalently coupled to cysteinefunctionalized phospholipids on the liposome surface for optical imaging of collagen.…”