title: Liposome-based Formulation for Intracellular Delivery of Functional Proteins.
abstract: The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Chatin
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Mével
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Devallière
authors.affiliation: CBMN UMR-CNRS 5248, Université de Bordeaux IPB, Pessac, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Dallet
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haudebourg
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France.
authors.forename: Pauline
authors.initials: P
authors.lastname: Peuziat
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France.
authors.forename: Thibault
authors.initials: T
authors.lastname: Colombani
authors.affiliation: UMR CNRS 6521, IFR 148 ScInBioS, Université de Bretagne Occidentale, Université Européenne de Bretagne, Brest, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Berchel
authors.affiliation: CBMN UMR-CNRS 5248, Université de Bordeaux IPB, Pessac, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Lambert
authors.affiliation: Faculté de médecine Paris-Descartes, INSERM U845, Paris, France.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Edelman
authors.affiliation: 1] Unité INSERM UMR 1087, CNRS UMR 6291, Nantes, France [2] Université de Nantes, L'institut du Thorax, Nantes, France [3] Present address: InCellArt, Nantes, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Pitard