Final Report Summary - DIRECTDELIVERY (Controlled fusion of liposomes and cells: a new pathway for direct drug delivery)
Fusion of lipid bilayers in cells facilitates the active transport of chemicals. Non-viral membrane fusion is regulated by a cascade of proteins as the process is highly regulated both in space and time. In eukaryotic cells, the so-called SNARE protein complex is at the heart of fusion. According to 2013 Nobel Prize laureate Sűdhof, how SNARE proteins promote fusion remains a major question in cell biology. This is because SNAREs are large, complex, membrane bound and therefore hard to handle, to modify and to manipulate. Inspired by SNARE-mediated fusion, I developed a model system able to induce targeted fusion.
The model system consists of a complementary pair of lipidated peptides able to form a heterodimeric coiled coil motif at the membrane interface similar to natural SNAREs. The mechanism of membrane fusion was studied using biophysical techniques, with an emphasis on peptide-peptide and peptide-lipid interactions. Furthermore, membrane fusion between liposomes and live cells was studied using optimized model systems.
The aims of this project were:
1) Understand the process of the peptide-controlled fusion of two membranes at the atomic, molecular and mesoscopic level.
2) Developing a new generic method for the controlled delivery of any (bio)molecule directly into the cytoplasm of a cell thereby omitting endocytotic pathways.
In the period covered by this project the following results have been obtained:
Aim 1: We studied the influence of the peptide sequence on the fusion of liposomes. A series of lipidated peptides were synthesized and a clear influence of the sequence on the rate of fusion was observed. We also varied the length of the spacer and its composition and a clear influence of the sequence on the rate of fusion was observed. The same was observed when we varied the lipid anchor (i.e. the transmembrane domain). We now have a library of lipidated peptides and understanding the mechanism underlying the fusion events was studied by a variety of techniques (monolayer and supported lipid bilayer studies, cryo-TEM and AFM measurements). This resulted in a better understanding of the mechanism of membrane fusion using this model system.
Aim 2: For this aim several routes were investigated. In the first approach we modified the cell membrane of living cells simply by the addition of an optimized lipidated peptide which assemble into micelles in the medium. These peptides bind/incroporate within minutes to the cell membrane as shown by fluorescence optical microscopy/confocal laser scanning microscopy. It has been shown that the membrane located peptides are still functional and are able to bind its complementary peptide resulting in the formation of coiled-coil complexes. In this manner it was possible to bind liposomes to a cell. In the last period we were also able to proof actual fusion between a cell and liposomes resulting in direct drug delivery.
In a second (alternative/spin-off) approach we modified the coiled-coil forming peptides with a large hydrophobic polypeptide block resulting in the formation of a wide variety of assemblies (i.e. polymersomes, bicelles, nanoparticles) depending on the exact composition and method of assembly preparation. In a pilot study, these materials showed to be useful as an adjuvant for sub-unit flu vaccine delivery in mice. These initial results were published, as well as a followup study showing that an optimized second generation of polypeptide-block-peptide assemblies were significantly more effective compared to commercial adjuvants.
The model system consists of a complementary pair of lipidated peptides able to form a heterodimeric coiled coil motif at the membrane interface similar to natural SNAREs. The mechanism of membrane fusion was studied using biophysical techniques, with an emphasis on peptide-peptide and peptide-lipid interactions. Furthermore, membrane fusion between liposomes and live cells was studied using optimized model systems.
The aims of this project were:
1) Understand the process of the peptide-controlled fusion of two membranes at the atomic, molecular and mesoscopic level.
2) Developing a new generic method for the controlled delivery of any (bio)molecule directly into the cytoplasm of a cell thereby omitting endocytotic pathways.
In the period covered by this project the following results have been obtained:
Aim 1: We studied the influence of the peptide sequence on the fusion of liposomes. A series of lipidated peptides were synthesized and a clear influence of the sequence on the rate of fusion was observed. We also varied the length of the spacer and its composition and a clear influence of the sequence on the rate of fusion was observed. The same was observed when we varied the lipid anchor (i.e. the transmembrane domain). We now have a library of lipidated peptides and understanding the mechanism underlying the fusion events was studied by a variety of techniques (monolayer and supported lipid bilayer studies, cryo-TEM and AFM measurements). This resulted in a better understanding of the mechanism of membrane fusion using this model system.
Aim 2: For this aim several routes were investigated. In the first approach we modified the cell membrane of living cells simply by the addition of an optimized lipidated peptide which assemble into micelles in the medium. These peptides bind/incroporate within minutes to the cell membrane as shown by fluorescence optical microscopy/confocal laser scanning microscopy. It has been shown that the membrane located peptides are still functional and are able to bind its complementary peptide resulting in the formation of coiled-coil complexes. In this manner it was possible to bind liposomes to a cell. In the last period we were also able to proof actual fusion between a cell and liposomes resulting in direct drug delivery.
In a second (alternative/spin-off) approach we modified the coiled-coil forming peptides with a large hydrophobic polypeptide block resulting in the formation of a wide variety of assemblies (i.e. polymersomes, bicelles, nanoparticles) depending on the exact composition and method of assembly preparation. In a pilot study, these materials showed to be useful as an adjuvant for sub-unit flu vaccine delivery in mice. These initial results were published, as well as a followup study showing that an optimized second generation of polypeptide-block-peptide assemblies were significantly more effective compared to commercial adjuvants.