Frank Versluis

Shape and release control of a peptide decorated vesicle through pH sensitive orthogonal supramolecular interactions.

A pH sensitive carrier is obtained by coating a cyclodextrin vesicle with an adamantane-terminated octapeptide through the formation of an inclusion complex. Upon lowering the pH from 7.4 to 5.0, the formation of peptide beta-sheets on the vesicle surface induces a transition of the bilayer from a sphere to a fiber. This transition is fully reversible and repeatable. The vesicles release their cargo upon fiber formation.

In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion

Complementary coiled coil forming lipidated peptides embedded in liposomal membranes are able to induce rapid, controlled, and targeted membrane fusion. Traditionally, such fusogenic liposomes are prepared by mixing lipids and lipidated peptides in organic solvent (e.g., chloroform). Here we prepared fusogenic liposomes in situ, i.e., by addition of a lipidated peptide solution to plain liposomes. As the lipid anchor is vital for the correct insertion of lipidated peptides into liposomal membranes, a small library of lipidated coiled coil forming peptides was designed in which the lipid structure was varied. The fusogenicity was screened using lipid and content mixing assays showing that cholesterol modified coiled coil peptides induced the most efficient fusion of membranes. Importantly, both lipid and content mixing experiments demonstrated that the in situ modification of plain liposomes with the cholesterol modified peptides yielded highly fusogenic liposomes. This work shows that existing membranes can be activated with lipidated coiled coil forming peptides, which might lead to highly potent applications such as the fusion of liposomes with cells.

In Vitro and In Vivo Supramolecular Modification of Biomembranes Using a Lipidated Coiled‐Coil Motif

The molecular building blocks available in biological systems self-assemble into defined structures in an extremely controlled manner. These structures must be flexible and adaptive to the environment in order to carry out their function in a regulated manner. Therefore, nature uses multiple weak interactions (e.g. hydrogen bonding and van der Waals interactions) to act as the glue to hold these structures together. When many weak interactions cooperatively combine, relatively stable entities are produced, which retain the ability to respond to external stimuli such as fluctuations in ion concentration, pH, and temperature. For many years, nature has been a source of inspiration for supramolecular chemistry. Scientists typically follow the bottom-up approach and design relatively simple molecules which assemble into functional materials with well-defined properties. Recent progress has resulted in molecular systems that are responsive to multiple stimuli and are therefore highly controlled, emulating nature ever more closely. A relatively new development is the application of supramolecular constructs in in vitro and in vivo environments to directly study and influence biological processes in live cells. Chemically tailored systems can be integrated into cell membranes, for example. This enables the modification or regulation of cellular behavior through external artificial signals. There are two approaches for introducing chemical species into a cell membrane by supramolecular chemistry: 1) specific binding of guest molecules to membrane-anchored biomolecules such as native proteins and 2) nonspecific labeling of membranes with the aid of hydrophobic and electrostatic interactions or through a chemical crosslinker. Lipidated peptides are particularly good candidates for application in biological systems as their aggregation behavior can be controlled by carefully balancing the hydrophobicity of the anchor and the hydrophilicity of the cargo; this aids the incorporation of lipidated peptides into membranes. Here we describe the use of a coiled-coil motif as the peptide segment, a highly specific recognition system that can be introduced into live cells. The coiled-coil motif acts as molecular Velcro and can thus be used to link distinct molecular constructs. An example of the specific labeling of proteins through coiled-coil formation was recently supplied by Matsuzaki et al. Surface modification through the nonspecific binding of polymers to cell membranes has also been studied, for example by Ijiro et al. Lipid-grafted polymers adhere to cell membranes and could potentially act as a scaffold to which a wide range of functional moieties could be attached, thereby intervening in the chemistry of the cell. Furthermore, cationic graft copolymers have also been shown to interact electrostatically with cell membranes, resulting in chemically altered cell membranes. Although these examples illustrate that in vitro membrane functionalization is a highly rewarding strategy, there are currently no examples of efficient in vivo strategies. Therefore, it is our goal to transiently modify lipid membranes through specific supramolecular interactions in in vitro and in vivo environments. For this purpose, we use a pair of complementary coiled-coil-forming lipidated peptides (E and K peptides) to specifically introduce a noncovalent and bio-orthogonal recognition motif to biological membranes (Scheme 1). Here, we describe a generic supramolecular tool which allows us to rapidly and efficiently form coiled-coil motifs at the surface of biological membranes. This is of interest as a wide range of molecular constructs can be introduced to the surface of the cell in this way. Coiled-coil-forming peptides E [(EIAALEK)3] and K [(KIAALKE)3] [12] were first covalently conjugated to PEG12 spacers (PEG= polyethylene glycol). Subsequently, a cholesterol moiety was coupled to the pegylated peptides yielding CPE and CPK (Scheme 1A). The cholesterol moiety allows for the immediate insertion of the lipidated peptides into membranes through hydrophobic interactions and the PEG12 moiety was incorporated to aid efficient molecular recognition between the peptide segments E and K. Recently we showed that upon the addition of micellar solutions of either CPE or CPK to plain liposomes, the lipidated peptides spontaneously inserted into liposomal membranes. In the current study, CHO cell membranes (CHO=Chinese hamster ovary) and the skin of zebrafish embryos were modified with coiled-coil-forming peptides by the addition of a micellar solution of CPE or CPK, resulting in immediate incorporation of these amphiphiles into the membranes. Subsequently, the complementary peptide was added, result[*] M. Sc. H. R. Zope, M. Sc. F. Versluis, Dr. J. Voskuhl, Dr. A. Kros Soft Matter Chemistry, Leiden Institute of Chemistry Leiden University P.O. Box 9502, 2300 RA Leiden (The Netherlands) E-mail: a.kros@chem.leidenuniv.nl

Synthetic Self-Assembled Materials in Biological Environments.

Synthetic self-assembly has long been recognized as an excellent approach for the formation of ordered structures on the nanoscale. Although the development of synthetic self-assembling materials has often been inspired by principles observed in nature (e.g., the assembly of lipids, DNA, proteins), until recently the self-assembly of synthetic molecules has mainly been investigated ex vivo. The past few years however, have witnessed the emergence of a research field in which synthetic, self-assembling systems are used that are capable of operating as bioactive materials in biological environments. Here, this up-and-coming field, which has the potential of becoming a key area in chemical biology and medicine, is reviewed. Two main categories of applications of self-assembly in biological environments are identified and discussed, namely therapeutic and imaging agents. Within these categories key concepts, such as triggers and molecular constraints for in vitro/in vivo self-assembly and the mode of interaction between the assemblies and the biological materials will be discussed.

Immobilization of liposomes and vesicles on patterned surfaces by a peptide coiled-coil binding motif.

Patchy surfaces: An azide-terminated self-assembled monolayer was patterned with the peptide sequence (EIAALEK)(3) by using microcontact printing. This sequence forms stable coiled-coil heterodimers with the complementary peptide (KIAALKE)(3). By introducing this peptide to the surface of phospholipid liposomes and cyclodextrin vesicles, liposomes and vesicles can be immobilized at the patterned surface.

Influence of pegylation on peptide-mediated liposome fusion

The effect of surface-attached PEG on the peptide-mediated fusion of liposomes was investigated. A complementary pair of coiled-coil forming lipidated peptides was introduced to two batches of small unilamellar liposomes separately. Upon mixing, efficient liposome membrane fusion was apparent when the liposomes were not decorated with pegylated lipids, however when the liposomes were pegylated the fusion was inhibited. A FRET-based fluorescence assay indicated that the fusion can be prevented effectively with less than two mole percent of pegylated lipid. DLS and CD spectroscopy were used to further evaluate the influence of pegylation on fusion. These data revealed that the pegylated lipids inhibit peptide complex formation and liposome docking, thereby preventing liposome fusion at the initial stage of the process. In contrast, when the PEG is not covalently attached to the liposome, no fusion inhibition was observed. Thus we conclude that the steric effect of the surface-bound PEG chains, which prevents sustained docking of liposomes, is the main cause of fusion inhibition.

A toolbox for controlling the properties and functionalisation of hydrazone-based supramolecular hydrogels

In recent years, we have developed a low molecular weight hydrogelator system that is formed in situ under ambient conditions through catalysed hydrazone formation between two individually non-gelating components. In this contribution, we describe a molecular toolbox based on this system which allows us to (1) investigate the limits of gel formation and fine-tuning of their bulk properties, (2) introduce multicolour fluorescent probes in an easy fashion to enable high-resolution imaging, and (3) chemically modify the supramolecular gel fibres through click and non-covalent chemistry, to expand the functionality of the resultant materials. In this paper we show preliminary applications of this toolbox, enabling covalent and non-covalent functionalisation of the gel network with proteins and multicolour imaging of hydrogel networks with embedded mammalian cells and their substructures. Overall, the results show that the toolbox allows for on demand gel network visualisation and functionalisation, enabling a wealth of applications in the areas of chemical biology and smart materials.

Negatively Charged Lipid Membranes Catalyze Supramolecular Hydrogel Formation

In this contribution we show that biological membranes can catalyze the formation of supramolecular hydrogel networks. Negatively charged lipid membranes can generate a local proton gradient, accelerating the acid-catalyzed formation of hydrazone-based supramolecular gelators near the membrane. Synthetic lipid membranes can be used to tune the physical properties of the resulting multicomponent gels as a function of lipid concentration. Moreover, the catalytic activity of lipid membranes and the formation of gel networks around these supramolecular structures are controlled by the charge and phase behavior of the lipid molecules. Finally, we show that the insights obtained from synthetic membranes can be translated to biological membranes, enabling the formation of gel fibers on living HeLa cells.

Coiled-coil driven membrane fusion: zipper-like vs. non-zipper-like peptide orientation.

Membrane fusion plays a central role in biological processes such as neurotransmission and exocytosis. An important class of proteins that induce membrane fusion are called SNARE (soluble N-ethyl malemeide sensitive factor attachment protein receptors) proteins. To induce membrane fusion, two SNARE proteins embedded in opposing membranes form a four-helix coiled-coil motif together with a third, cytoplasmic, SNARE protein. Coiled-coil formation brings the two membranes into close proximity allowing fusion to occur. Importantly, structural investigations have demonstrated that native membrane fusion only occurs when the orientation of the coiled-coil motif resembles that of a zipper. The zipper orientation arises when parallel coiled-coil formation takes place between peptides that are anchored into apposing membranes at identical termini, thereby forcing the membranes into close contact. Recently, we have designed a synthetic model for membrane fusion, which is based on a set of lipidated coiled-coil forming peptide pairs which are denoted E-K. When incorporated into liposomal membranes, coiled-coil formation between these lipidated peptides induces targeted and efficient membrane fusion of liposomes. Our model system mimics SNARE-driven membrane fusion, as it contains a coiled-coil motif which has a zipper-like orientation, similar to that of the SNARE proteins. Here we investigate whether the zipper-like orientation of the coiled-coil motifs is a prerequisite for membrane fusion in our model system. Our strategy is based on conjugation of the transmembrane anchor to either the N- or the C-terminus of peptides E and K. Whereas the use of a set of complementary peptides with the membrane anchor on identical peptide termini yields the zipper-like orientation of the coiled-coil complex, membrane anchors on opposite peptide termini results in a non-zipper-like coiled-coil orientation. Surprisingly, it was observed that efficient and targeted membrane fusion was induced even when the coiled-coil motif did not form the zipper-like orientation. This demonstrates that for our model system, the zipper model for membrane fusion does not apply.

Power struggles between oligopeptides and cyclodextrin vesicles

Stimulus responsive supramolecular assemblies were constructed by decorating the surface of β-cyclodextrin vesicles (CDVs) with β-sheet forming oligopeptides. Three octapeptides were used to coat the CDVs: adamantane-(leu-glu)4 (1), adamantane-PEG4-(leu-glu)4 (2) and (adamantane-PEG4)2-(leu-glu)4 (3). The adamantane moiety ensured the binding of the peptides at the surface of the CDVs. At pH 7.2, all the peptides typically adopt a random coil secondary structure. Upon acidification to ∼pH 5, however, a reversible transition to well-defined β-sheet structures was observed. The pH at which this transition occurred was found to be influenced by the PEG4-spacer and the number of adamantane residues conjugated to the octapeptide hybrids. The structural rearrangement of the peptide backbone was found to induce a morphological transition of the entire assembly. For the peptide (1)–CDV assembly a reversible transition to fiber like structures was observed, whereas bundles of aggregated fibers were present in the peptide (2)–CDV assembly. Upon adding peptide (3) to the CDVs, the structural integrity of the CDVs was lost. The peptide (1)–CDV assembly shows promise as a delivery device due to the pH-induced vesicle-to-fiber transition, resulting in a release of its cargo from the aqueous interior of the CDVs. The amount of released cargo could be controlled as a function of pH and peptide concentration.