Self-assembled Cyclic Peptide Research Articles

Self-assembly mechanisms of cyclic peptide nanotubes in the presence of water molecules; study of hydrogen bonding, aggregation and Mir-210 gene delivery

Self-assembling cyclic peptides are a type of bioinspired supramolecular building blocks. They have the ability to stack together, forming supramolecular cyclic peptide nanotubes. This stacking is driven by β-sheet-like hydrogen bonding. In general, a single hydrogen bond is not strong enough to form ordered supramolecular structures. However, when multiple hydrogen bonds are aligned and built into arrays, they exhibit increased strength and directionality. This enables the formation of well-defined and organized supramolecular structures. The continuous advancements in understanding cyclic peptides and their unique properties open new doors for their application in functional materials, ion transport channels, and drug delivery systems, including gene therapy. When it comes to gene therapy, which involves introducing genetic material into cells to treat or prevent diseases, delivery systems play a crucial role. The use of cyclic peptides in these delivery systems can enhance the stability, specificity, and cellular uptake of the therapeutic cargo, thereby improving the overall success of gene therapy treatments. Hydrogen bond formation makes the cyclic peptide nanotube more stable and has an important role in its formation. The number of hydrogen bonds between cyclic peptides is 117 on average during the simulation time of 200 ns. Also, hydrogen bonds formation between Mir-210 and cyclic peptide nanotube during the simulation time observed which is an average of 17 bonds.

Influence of salt and temperature on the self-assembly of cyclic peptides in water: a molecular dynamics study.

It is found in the literature that cyclic peptides (CPs) are able to self-assemble in water to form cyclic peptide nanotubes (CPNTs) and are used extensively in the field of nanotechnology. Several factors influence the formation and stability of these nanotubes in water. However, an extensive study of the contribution of several important factors is still lacking. The purpose of this study is to explore the effect of temperature and salt (NaCl) on the association tendency of CPs. Furthermore, the self-association behavior of CPs in aqueous solutions at various temperatures is also thoroughly discussed. Cyclo-[(Asp-D-Leu-Lys-D-Leu)2] is considered for this study and a series of classical molecular dynamics (MD) simulations at three different temperatures, viz. 280 K, 300 K, and 320 K, both in pure water and in NaCl solutions of different concentrations are carried out. The calculations of radial distribution functions, preferential interaction parameters, cluster formation and hydrogen bonding properties suggest a strong influence of NaCl concentration on the association propensity of CPs. Low NaCl concentration hinders CP association while high NaCl concentration facilitates the association of CPs. Besides this, the association of CPs is found to be enhanced at low temperature. Furthermore, the thermodynamics of CP association is predominantly found to be enthalpy driven in both the presence and absence of salt. No crossover between enthalpy and entropy in CP association is observed. In addition, the MM-GBSA method is used to investigate the binding free energies of the CP rings that self-assembled to form nanotube like structures at all three temperatures.

Cyclization and Self-Assembly of Cyclic Peptides.

Cyclic peptides are a fascinating class of molecules that can be programmed to fold or self-assemble into diverse mono- and multidimensional structures with potential applications in biomedicine, nanoelectronics, or catalysis. Herein we describe on-resin procedures to carry out head-to-tail peptide cyclization based on orthogonal protected linear structures. We also present essential characterization tools for obtaining dynamic and structural information, including the visualization cyclic peptide assembly into nanotubes (AFM, TEM) as well as the use of fluorescence microscopy.

Transmembrane Self-Assembled Cyclic Peptide Nanotubes Based on α-Residues and Cyclic δ-Amino Acids: A Computational Study.

Self-assembling cyclic peptide nanotubes have been shown to function as synthetic, integral transmembrane channels. The combination of natural and nonnatural aminoacids in the sequence of cyclic peptides enables the control not only of their outer surface but also of the inner cavity behavior and properties, affecting, for instance, their permeability to different molecules including water and ions. Here, a thorough computational study on a new class of self-assembling peptide motifs, in which δ-aminocycloalkanecarboxylic acids are alternated with natural α-amino acids, is presented. The presence of synthetic δ-residues creates hydrophobic regions in these α,δ-SCPNs, which makes them especially attractive for their potential implementation in the design of new drug or diagnostic agent carrier systems. Using molecular dynamics simulations, the behavior of water molecules, different ions (Li+, Na+, K+, Cs+, and Ca2+), and their correspondent counter Cl− anions is extensively investigated in the nanoconfined environment. The structure and dynamics are mutually combined in a diving immersion inside these transmembrane channels to discover a fascinating submarine nanoworld where star-shaped water channels guide the passage of cations and anions therethrough.

Effect of Water Models on Transmembrane Self-Assembled Cyclic Peptide Nanotubes.

Self-assembling cyclic peptide nanotubes can form nanopores when they are inserted in lipid bilayers, acting as ion and/or water permeable channels. In order to improve the versatility of these systems, it is possible to specifically design cyclic peptides with a combination of natural and non-natural amino acids, enabling the control of the nature of the inner cavity of the channels. Here, the behavior of two types of self-assembling peptide motifs, alternating α-amino acids with γ- or δ-aminocycloalkanecarboxylic acids, is studied via molecular dynamics (MD) simulations. The behavior of water molecules in nanopores is expected to affect the properties of these channels and therefore merits detailed examination. A number of water models commonly used in MD simulations have been validated by how well they reproduce bulk water properties. However, it is less clear how these water models behave in the nanoconfined condition inside a channel. The behavior of four different water models—TIP3P, TIP4P, TIP4P/2005, and OPC—are evaluated in MD simulations of self-assembled cyclic peptide nanotubes of distinct composition and diameter. The dynamic behavior of the water molecules and ions in these designed artificial channels depends subtly on the water model used. TIP3P water molecules move faster than those of TIP4P, TIP4P/2005, and OPC. This demeanor is clearly observed in the filling of the nanotube, in water diffusion within the pore, and in the number and stability of hydrogen bonds of the peptides with water. It was also shown that the water model influences the simulated ion flux through the nanotubes, with TIP3P producing the greatest ion flux. Additionally, the two more recent models, TIP4P/2005 and OPC, which are known to reproduce the experimental self-diffusion coefficient of bulk water quite well, exhibit very similar results under the nanoconfined conditions studied here. Because none of these models have been parametrized specifically for waters confined in peptide nanotubes, this study provides a point of reference for further validation.

Membrane targeting antimicrobial cyclic peptide nanotubes – an experimental and computational study

The search of new antibiotics, particularly with new mechanisms of action, is nowadays a very important public health issue, due to the worldwide increase of resistant pathogens. Within this effort, much research has been done on antimicrobial peptides, because having the membrane as a target, they represent a new antibiotic paradigm. Among these, cyclic peptides (CPs) made of sequences of D- and L-amino acids have emerged as a new class of potential antimicrobial peptides, due to their expected higher resistance to protease degradation. These CPs are planar structures that can form Self-assembled Cyclic Peptide Nanotubes (SCPNs), in particular in the presence of lipid membranes. Aiming at understanding their mechanism of action, we used biophysical experimental techniques (DSC and ATR-FTIR) together with Coarse-grained molecular dynamics (CG-MD) simulations, to characterize the interaction of these CPs with model membranes of different electrostatic charges’ contents. DSC results revealed that the CPs show a strong interaction with negatively charged membranes, with differences in the strength of interactions depending on peptide and on membrane charge content, at odds with no or mild interactions with zwitterionic membranes. ATR-FTIR suggested that the peptides self-assemble at the membrane surface, adopting mainly a β-structure. The experiments with polarized light showed that in most cases they lie parallel to the membrane surface, but other forms and orientations are also apparent, depending on peptide structure and lipid:peptide ratio. The nanotube formation and orientation, as well as the dependence on membrane charge were also confirmed by the CG-MD simulations. These provide detail on the position and interactions, in agreement with the experimental results. Based on the findings reported here, we could proceed to the design and synthesis of a second-generation CPs, based on CP2 (soluble peptide), with increased activity and reduced toxicity.

Hypothesis: daptomycin permeabilizes membranes by forming self assembled nanotubes

Daptomycin is the only lipopeptide antibiotic that is widely used in clinical practice. It was discovered by Eli Lilly and then studied and commercialized by Cubist Pharmaceuticals in 2003. Although this antibiotic has been used for 17 years, the debate over its mechanism of action is ongoing. In this paper, we discuss the different hypotheses on the mode of action of this antibiotic with a primary focus on the bacterial membrane permeabilization as the main mechanism of action. By comparing the experimental data on the oligomerization of daptomycin in membranes with properties of self-assembling cyclic peptides, we conclude that the structure of daptomycin oligomer should resemble the structures of peptide nanotubes that serve as ion channels in membranes.

Energy behaviour of Doxorubicin interacting with peptide nanotubes

Self-assembling cyclic peptide nanotubes are widely investigated in many scientific fields due to many of their properties; controllable sizes, stable, and simple formation. In this research, the cyclic peptide nanotubes are modelled as the carriers to interact with a drug molecule. On employing the Lennard-Jones function and the continuous approximation, the energy value of the system can be evaluated. The main outcome of this research is a mathematical expression describing the relation of energy, radii of drug and peptide and the spacing between peptide units. We find that the drug is most stable when it is in the spacing between subunits of the peptide nanotubes. We also consider the energy behaviour of an offset drug inside three different sizes of the cyclic peptide nanotubes. We predict that the drug can be encapsulated inside the peptide nanotube when the difference of their radii is around 3 A.

1D to 2D Self Assembly of Cyclic Peptides.

Despite recent developments in two-dimensional self-assembly, most supramolecular 2D materials are assembled by tedious methodologies, with complex surface chemistry and small sizes. We here report d/l-alternating cyclic peptides that undergo one-dimensional self-assembly into amphiphilic nanotubes, which subsequently arrange as tubular bilayers to form giant nanosheets in the mesoscale. Reversible transitions between the assembled, dispersed, and aggregated states of these nanosheets can be triggered by external stimuli. The characteristic flexibility, defined chemical topology, and length scale of these nanosheets set a clear distinction between this new supramolecular architecture and previously reported 2D nanostructures. The sequential 1D-to-2D self-assembly of peptides described here provides a conceptually new approach to achieve two-dimensional materials with hierarchical organization. These giant nanosheets represent one of the largest 2D supramolecular materials ever made, with potential application as long-range molecular transporters, responsive surfaces, and (bio)sensors.

Competitive double-switched self-assembled cyclic peptide nanotubes: a dual internal and external control.

"Intelligent" materials based on synthetic small molecules that become functional only under specific conditions provide new opportunities for developing regulated systems aimed at a large number of applications. For instance, biologically active supramolecular entities that are sensitive to environmental conditions, such as the presence of bacterial membranes, are extremely interesting in biomedicine. In this work, we have designed and investigated, using molecular dynamics simulations, a doubly modulable nanotube formed by the self-assembly of cyclic peptides sensitive to both the presence of a lipid membrane and the pH of the aqueous media. The cyclic peptides were designed to self-assemble into peptide nanotubes in the presence of a lipid bilayer and at low pH values. Under these conditions, the residual side chains point outside the cyclic peptides, being exposed to the lipid bilayer, and the inner groups (carboxylic acids) are protonated, thus allowing the permeation of water and preventing that of ions. Higher pH values are expected to create carboxylate groups at the lumen of the peptides, leading to the disassembly of the nanotube, the attraction and translocation of ions towards the hydrophobic core of the bilayer, and eventually killing the target malignant cells. Our results suggest that by introducing a second switch in a membrane sensitive system, it is possible to modulate its interaction with the lipid bilayer. This opens the door to new strategies for the preparation of antimicrobial peptides that interact at the membrane level.

Systematic study of the structural parameters affecting the self-assembly of cyclic peptide-poly(ethylene glycol) conjugates.

Self-assembling cyclic peptides (CP) consisting of amino acids with alternating d- and l-chirality form nanotubes by hydrogen bonding, hydrophobic interactions, and π-π stacking in solution. These highly dynamic materials are emerging as promising supramolecular systems for a wide range of biomedical applications. Herein, we discuss how varying the polymer conformation (linear vs. brush), as well as the number of polymer arms per peptide unimer affects the self-assembly of PEGylated cyclic peptides in different solvents, using small angle neutron scattering. Using the derived information, strong correlations were drawn between the size of the aggregates, solvent polarity, and its ability to compete for hydrogen bonding interactions between the peptide unimers. Using these data, it could be possible to engineer cyclic peptide nanotubes of a controlled length.

Insight of Transmembrane Processes of Self-Assembling Nanotubes Based on a Cyclic Peptide Using Coarse Grained Molecular Dynamics Simulation.

Transmembrane self-assembling cyclic peptide (SCP) nanotubes are promising candidates for delivering specific molecules through cell membranes. The detailed mechanisms behind the transmembrane processes, as well as stabilization factors of transmembrane structures, are difficult to elucidate through experiments. In this study, the effects of peptide sequence and oligomeric state on the transmembrane capabilities of SCP nanotubes and the perturbation of embedded SCP nanotubes acting on the membrane were investigated based on coarse grained molecular dynamics simulation. The simulation results reveal that hydrophilic SCP oligomers result in the elevation of the energy barrier while the oligomerization of hydrophobic SCPs causes the reduction of the energy barrier, further leading to membrane insertion. Once SCP nanotubes are embedded, membrane properties such as density, thickness, ordering state and lateral mobility are adjusted along the radial direction. This study provides insight into the transmembrane strategy of SCP nanotubes and sheds light on designing novel transport systems.

Self-Assembling Molecular Capsules Based on α,γ-Cyclic Peptides.

A new capsule based on a β-sheet self-assembling cyclic peptide with the ability to recognize and release several guests is described. The host structure is composed of two self-complementary α,γ-cyclic peptides bearing a Zn porphyrin cap that is used for the selective recognition of the guest. The two components are linked through two dynamic covalent bonds. The combination of binding forces, including hydrogen bonding, metal coordination, and dynamic hydrazone bonds, allows the reversible recognition of long bipyridine guests. The affinity for these ligands showed a strong dependence on the guest length. Delivery of the encapsulated ligand can be achieved by hydrolysis of hydrazones to disrupt the sandwich complex structure.

Recent advances in controlling the internal and external properties of self-assembling cyclic peptide nanotubes and dimers.

One of the most powerful strategies for the preparation of nanotubes is based on the stacking of flat-shaped cyclic peptide components. This strategy allows precise control of the nanotube internal diameter, the external properties and, more recently, the structural characterisitics of the internal cavity. The recent advances in these technologies and the applications of the resulting materials are described.

Study of (Cyclic Peptide)-Polymer Conjugate Assemblies by Small-Angle Neutron Scattering.

We present a fundamental study into the self-assembly of (cyclic peptide)-polymer conjugates as a versatile supramolecular motif to engineer nanotubes with defined structure and dimensions, as characterised in solution using small-angle neutron scattering (SANS). This work demonstrates the ability of the grafted polymer to stabilise and/or promote the formation of unaggregated nanotubes by the direct comparison to the unconjugated cyclic peptide precursor. This ideal case permitted a further study into the growth mechanism of self-assembling cyclic peptides, allowing an estimation of the cooperativity. Furthermore, we show the dependency of the nanostructure on the polymer and peptide chemical functionality in solvent mixtures that vary in the ability to compete with the intermolecular associations between cyclic peptides and ability to solvate the polymer shell.

Glucose derivatives substitution and cyclic peptide diameter effects on the stability of the self-assembled cyclic peptide nanotubes; a joint QM/MD study

Dynamical behavior and the stability of eighteen nanostructures composed of cyclic peptide (CP) with the general structure of the cyclo(CO(CH2)n=4, 6, 10COCyst), in the gas phase, water and chloroform were investigated during 50ns molecular dynamic (MD) simulations. CP dimers and cyclic peptide nanotubes (CPNTs) are more stable in chloroform than water and this stability is reversely correlated with the ring size of the CP units. Also the effect of glucose derivatives substitution, d-glucose (S1) and N-methyl-d-glucamine (S2), on the stability and other physicochemical properties of the CP dimers and CPNTs were evaluated. These substitutions increase the inner-subunits hydrogen bonds (H-bond) which in turn increase the stability of these structures. Moreover, the S2 substitution in comparison to the S1 makes dimers and CPNTs more stable. Gibbs free energy analysis based on the MM-PBSA and MM-GBSA calculations confirmed that substitutions affect the stability of the studied nanostructures, considerably and an increase in the length of the CPNT units reduces their stability. Quantum chemistry calculations on the dimer structures using the density functional theory (DFT) and DFT-D3 methods were performed. Based on the DFT-D3 calculations, it was revealed that the dispersion interactions play a key role in the dimerization process. The ring size increment, elevates the dispersion interaction energy which is accordance with the MD results. H-bond formation between the CO and NH groups of the CP units inside the dimers have been analyzed by using the quantum theory of atoms in molecules and natural bond orbital description. Finally, through these analyses, the electrostatic interaction between the mentioned groups have been evaluated.

Self-assembling α,γ-cyclic peptides that generate cavities with tunable properties.

The design and synthesis of β-sheet-based self-assembling cyclic peptides with tunable cavities is described. The incorporation of a γ-amino acid with a hydroxyl group at C2 allows the incorporation of different groups that modify the internal properties of the resulting dimeric ensemble. These dimers can entrap different guests depending on the properties of the group at C2. The guest defines the geometry of the resulting aggregate.

Modelling ion, water and ion-water cluster entering peptide nanotubes

Recently, organic nanostructures have attracted much attention, and amongst them peptide nanotubes are of interest in many fields of application including medicine and nanobiotechnology. Peptide nanotubes are formed by self-assembly of cyclic peptides with alternating L- and D-amino acids. Due to their biodegradability, flexible design and easy synthesis, many applications have been proposed such as artificial transmembrane ion channels, templates for nanoparticles, mimicking pore structures, nanoscale testing tubes, biosensors and carriers for targeted drug delivery. The mechanisms of an ion, a water molecule and an ion–water cluster entering into a peptide nanotube of structure cyclo[(-D-Ala-L-Ala-)4] are explored here. In particular, the Lennard-Jones potential and a continuum approach are employed to determine three entering mechanisms: (i) through the tube open end, (ii) through a region between each cyclic peptide ring and (iii) around the edge of the tube open end. The results show that while entering the nanotube by method (i) is possible, an ion or a molecule requires initial energy to overcome an energetic barrier to be able to enter the nanotube through positions (ii) and (iii). Due to its simple structure, the D-, L-Ala cyclopeptide nanotube is chosen in this model; however, it can be easily extended to include more complicated nanotubes. doi:10.1017/S1446181115000164

MODELLING ION, WATER AND ION–WATER CLUSTER ENTERING PEPTIDE NANOTUBES

Recently, organic nanostructures have attracted much attention, and amongst them peptide nanotubes are of interest in many fields of application including medicine and nanobiotechnology. Peptide nanotubes are formed by self-assembly of cyclic peptides with alternating L- and D-amino acids. Due to their biodegradability, flexible design and easy synthesis, many applications have been proposed such as artificial transmembrane ion channels, templates for nanoparticles, mimicking pore structures, nanoscale testing tubes, biosensors and carriers for targeted drug delivery. The mechanisms of an ion, a water molecule and an ion–water cluster entering into a peptide nanotube of structure cyclo[(-D-Ala-L-Ala-)$_{4}$] are explored here. In particular, the Lennard-Jones potential and a continuum approach are employed to determine three entering mechanisms: (i) through the tube open end, (ii) through a region between each cyclic peptide ring and (iii) around the edge of the tube open end. The results show that while entering the nanotube by method (i) is possible, an ion or a molecule requires initial energy to overcome an energetic barrier to be able to enter the nanotube through positions (ii) and (iii). Due to its simple structure, the D-, L-Ala cyclopeptide nanotube is chosen in this model; however, it can be easily extended to include more complicated nanotubes.

In Vitro and In Vivo Efficacy of Self-Assembling RGD Peptide Amphiphiles for Targeted Delivery of Paclitaxel

The objective of this work was to compare the efficacy of self-assembling cyclic and linear RGD peptide amphiphiles as carriers for delivering paclitaxel to αvβ3 integrin overexpressing tumors. Linear (C18-ADA5-RGD) and cyclic (C18-ADA5-cRGDfK) peptide amphiphiles were synthesized and characterized for CMC, aggregation number and micelle stability using fluorescence spectroscopy methods. Size and morphology of micelles was studied using TEM. Fluorescence polarization and confocal microscopy assays were established to compare binding and internalization of micelles. The targeting efficacy was studied in A2058 cells using cytotoxicity assay as well as in vivo in melanoma xenograft mouse model. The linear and cyclic RGD amphiphiles exhibited CMC of 25 and 8 μM, respectively, formed nano-sized spherical micelles and showed competitive binding to αvβ3 integrin protein. FITC-loaded RGD micelles rapidly internalized into A2058 melanoma cells. Paclitaxel-loaded RGD micelles exhibited higher cytotoxicity compared with free drug in A2058 cells in vitro as well as in vivo. Cyclic RGD micelles exhibited better targeting efficacy but were less effective compared to linear RGD micelles as drug delivery vehicle due to lower drug solubilization capacity and lesser kinetic stability. Results from the study proved the effectiveness of self-assembling low molecular weight RGD amphiphiles as carriers for targeted delivery of paclitaxel.