title: Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger.
abstract: Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.
authors.affiliation: Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
authors.forename: E
authors.identifier: 0000-0002-0628-3350
authors.initials: E
authors.lastname: Oude Blenke
authors.affiliation: Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Sleszynska
authors.affiliation: Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
authors.forename: M J W
authors.initials: MJ
authors.lastname: Evers
authors.affiliation: Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Storm
authors.affiliation: Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
authors.forename: N I
authors.initials: NI
authors.lastname: Martin
authors.affiliation: Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Mastrobattista
mesh_terms: D000595:Amino Acid Sequence; D057930:Click Chemistry; D003692:Delayed-Action Preparations / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006835:Hydrazones / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D008555:Melitten / Q000737:chemistry / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: C519184:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000); D003692:Delayed-Action Preparations; D006835:Hydrazones; D008081:Liposomes; D008301:Maleimides; D010714:Phosphatidylethanolamines; D013438:Sulfhydryl Compounds; D008555:Melitten; C043592:maleimide; D011092:Polyethylene Glycols