Harshal R. Zope

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

Coiled-coil peptide motifs as thermoresponsive valves for mesoporous silica nanoparticles.

Coiled-coil peptide motifs were used as thermo-responsive valves for mesoporous silica nanoparticles (MSNs). The controlled release of a model drug as a function of temperature was demonstrated.

Polymer-induced liquid precursor (PILP) phases of calcium carbonate formed in the presence of synthetic acidic polypeptides—relevance to biomineralization

Polymer-induced liquid precursor (PILP) phases of calcium carbonate have attracted significant interest due to possible applications in materials synthesis, and their resemblance to intermediates seen in biogenic mineralisation processes. Further, these PILP phases have been formed in vitro using polyelectrolytes such as poly(aspartic acid) which bears many structural parallels to the highly acidic biomacromolecules that are associated with biogenic calcium carbonate. This article describes experiments which investigate how the composition of acidic polypeptides determines their ability to form PILP phases of CaCO3, and therefore whether it is feasible that the acidic biomacromolecules extracted from CaCO3 biominerals could also function in this way. A series of random copoly(amino acid)s constructed from 80–20%, 50–50% and 20–80% aspartic acid and serine residues were synthesised and their effect on CaCO3 precipitation was determined. A strong correlation between the composition and function of the polypeptide was observed. Only the polypeptide containing 80% aspartic acid residues (Asp80%–Ser20%) induced the formation of continuous CaCO3 films, which provide a fingerprint of an intermediary PILP phase, while addition of Mg2+ also facilitated the formation of expanded film-like structures with the polypeptide Asp50%–Ser50%. In contrast, the weakly-acidic polypeptide Asp20%–Ser80% had only a minor effect on the crystal morphologies and also failed to aid infiltration of CaCO3 into small pores. These results therefore demonstrate that counter-ion induced phase separation of highly acidic biomacromolecules proteins appears to be entirely feasible based upon their composition, but that evidence for the operation of this mineralisation mechanism in vivo is still required.

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.

Polymersomes enhance the immunogenicity of influenza subunit vaccine

In this study poly(γ-benzyl-L-glutamate)-K (PBLG50-K) polymersomes are tested as an immune adjuvant for an antigen, influenza hemagglutinin (HA). The polymersomes were prepared according to a solvent removal method and loaded with HA antigensvia adsorption. The immunogenicity of the resulting hybrid assemblies was tested in vivo, resulting in an improvement of the immune response for the influenza antigen co-administered with the polymersomes.

Library of Random Copolypeptides by Solid Phase Synthesis

Random copolypeptides are promising and versatile bioinspired macromolecules of minimal complexity for studying their interactions with both living and synthetic matter. They provide the opportunity to investigate the role of, for example, total net charge and hydrophobicity through simply changing the monomer composition, without considering the effect of specific sequences or secondary structure. However, synthesizing large libraries of these polymers so far was prohibited by the time-consuming preparation methods available (ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides and enzymatic polymerization of amino acids). Here we report the automated solid phase synthesis (SPS) of a complete library of polypeptides containing Glu, Lys, and Ala monomers with excellent control over the degree of polymerization and composition and with polydispersity indices (PDIs) between 1.01 and 1.001, which is impossible to achieve by other methods. This method provides access to a library of polymers with a precisely defined total charge that can range from approximately -15 to +15 per chain and with a disordered conformation almost completely devoid of any secondary structure. In solution the polymers are largely present as unimers, with only the most hydrophobic polypeptides showing slight signs of aggregation. Our new approach provides convenient access to libraries of this versatile class of polymers with tunable composition, which can be used in a wide variety of physicochemical studies as a tool that allows systematic variation of charge and hydrophobicity, without the interference of secondary structure or aggregation on their performance.

Determination of oligomeric states of peptide complexes using thermal unfolding curves

In their native form peptides are often found as oligomeric complexes, meaning they consist of more than one peptide chain. Coiled coils and helical bundles are common examples of such complexes. Their oligomeric state needs to be known precisely as this tremendously influences their biochemical and biophysical properties. The extensive analysis of circular dichroism spectroscopic data is commonly used to investigate the thermodynamics of binding and folding of these complexes. Here we present FitDis! an easy‐to‐use programme, which fits the most common two‐state unfolding transition to the measured thermal unfolding curves of any oligomer of any stoichiometry. We demonstrate, with simulated and real examples, that the comparison of different stoichiometric models fitted to the same dataset reveals the oligomeric states of these complexes along with detailed thermodynamic information. This method will significantly ease the analysis of and increase the amount of information gained from, the thermal unfolding curves of peptide complexes.

Peptide Amphiphile Nanoparticles Enhance the Immune Response Against a CpG‐Adjuvanted Influenza Antigen

Cationic peptide amphiphile nanoparticles are employed for co-delivery of immune modulator CpG and antigen. This results in better targeting to the antigen presenting cells and eliciting strong Th1 response, which is effective against the intracellular pathogens.