Amphipathic design dictates self-assembly, cytotoxicity and cell uptake of arginine-rich surfactant-like peptides.

Amphiphilicity is the most critical parameter in the self-assembly of surfactant-like peptides (SLPs), regulating the way by which hydrophobic attraction holds peptides together. Its effects go beyond supramolecular assembly and may also trigger different cell responses of bioactive peptide-based nanostructures. Herein, we investigate the self-assembly and cellular effects of nanostructures based on isomeric SLPs composed by arginine (R) and phenylalanine (F). Two amphipathic designs were studied: a diblock construct F4R4 and its bolaamphiphile analog R2F4R2. A strong sequence-dependent polymorphism emerges with appearance of globules and vesicle-like assemblies, or flat nanotapes and cylindrical micelles. The diblock construct possesses good cell penetrating capabilities and effectiveness to kill SK-MEL-28 melanoma tumor cells, in contrast to reduced intracellular uptake and low cytotoxicity exhibited by the bolaamphiphilic form. Our findings demonstrate that amphipathic design is a relevant variable for self-assembling SLPs to modulate different cellular responses and may assist in optimizing the production of nanostructures based on arginine-enriched sequences in cell penetrating and antimicrobial peptides.

Cellulose elementary fibril orientation in the spruce S1-2 transition layer.

The tight organization of major wood cell wall polymers limits the swellability, solubility and reactivity of cellulose fibers during the production of regenerated textile fibers, nanocellulose, bioethanol, and many other value-added products. However, the ultrastructural assembly of cellulose elementary fibrils (EF) and matrix materials in one of the outer layers, i.e. S1-2 transition layer of wood cell wall, is far from being understood. Here, single-axis electron tomography on ultrathin spruce sections was applied to observe the three-dimensional (3D) structure of the S1-2 layer. The nanoscale geometries of the EFs were further quantitatively modeled through mathematical fitting of the tomographic subvolumes by suitable parametric space curves. The results showed that crisscross, bundled and parallel EF organizations are all present in this layer; the former two exhibit a denser structure. Several quantitative measures such as distances and angles were obtained for the analyzed structures. The result obtained in this study suggests that the S1-2 transition layer differs in structure than the principal cell wall layers. The structural differences and its possible role in wood cell wall have been discussed. These results will enhance our understanding of the swellability, accessibility and solubility of woody biomass for its conversion into the aforementioned value-added products.

The Effect of Lipidation on the Self-Assembly of the Gut-Derived Peptide Hormone PYY3-36.

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3-36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3-36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the ß-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3-36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3-36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity.

Self-Assembly of Telechelic Tyrosine End-Capped PEO Star Polymers in Aqueous Solution.

We investigate the self-assembly of two telechelic star polymer-peptide conjugates based on poly(ethylene oxide) (PEO) four-arm star polymers capped with oligotyrosine. The conjugates were prepared via N-carboxy anhydride-mediated ring-opening polymerization from PEO star polymer macroinitiators. Self-assembly occurs above a critical aggregation concentration determined via fluorescence probe assays. Peptide conformation was examined using circular dichroism spectroscopy. The structure of self-assembled aggregates was probed using small-angle X-ray scattering and cryogenic transmission electron microscopy. In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates that show self-assembly into ß-sheet fibrils, the star architecture suppresses fibril formation and micelles are generally observed instead, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These systems present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.

Sequence length dependence in arginine/phenylalanine oligopeptides: Implications for self-assembly and cytotoxicity.

We present a detailed study on the self-assembly and cytotoxicity of arginine-rich fragments with general form [RF]n (n=1-5). These highly simplified sequences, containing only two l-amino acids, provide suitable models for exploring both structure and cytotoxicity features of arginine-based oligopeptides. The organization of the sequences is revealed over a range of length scales, from the nanometer range down to the level of molecular packing, and their cytotoxicity toward C6 rat glioma and RAW264.7 macrophage cell lines is investigated. We found that the polymorphism is dependent on peptide length, with a progressive increase in crystalline ordering upon increasing the number of [RF] pairs along the backbone. A dependence on length was also found for other observables, including critical aggregation concentrations, formation of chiral assemblies and half maximum inhibitory concentrations (IC50). Whereas shorter peptides self-assemble into fractal-like aggregates, clear fibrillogenic capabilities are identified for longer sequences with octameric and decameric chains exhibiting crystalline phases organized into cross-ß structures. Cell viability assays revealed dose-dependent cytotoxicity profiles with very similar behavior for both glioma and macrophage cell lines, which has been interpreted as evidence for a nonspecific mechanism involved in toxicity. We propose that structural organization of [RF]n peptides plays a paramount role regarding toxicity due to strong increase of local charge density induced by self-assemblies rich in cationic groups when interacting with cell membranes.

Cellulose nanocrystals by acid vapour: towards more effortless isolation of cellulose nanocrystals.

Cellulose nanocrystals (CNCs) are topical in materials science but their full potential is yet to be fulfilled because of bottlenecks in the production: the process consumes huge amounts of water, recycling the strong acid catalyst is difficult, and purification steps are cumbersome, particularly with lengthy dialysis. Production of CNCs with HCl vapour overcomes many of these difficulties but the dispersion of CNCs from the already hydrolysed fibre matrix is a formidable challenge. This study is a fundamental effort to explore very basic means to facilitate CNC dispersion from cotton linter fibres (filter paper), hydrolysed to levelling off degree of polymerization by HCl vapour. The introduction of carboxylic groups on the cellulose crystal surface proved the most efficient method to alleviate dispersion with good yields (ca. 50%) and a provisional possibility to tune the CNC length. By contrast, attempts to directly disperse untreated hydrolysed fibres in various organic solvents and aqueous surfactant solutions were unsuccessful. The results showed that hydrolysis of native cellulose fibres by HCl vapour is indeed a viable method for producing CNCs but it has more potential as a pre-treatment step rather than a full-fledged process on its own.

The Origin of Hierarchical Structure Formation in Highly Grafted Symmetric Supramolecular Double-Comb Diblock Copolymers.

Involving supramolecular chemistry in self-assembling block copolymer systems enables design of complex macromolecular architectures that, in turn, could lead to complex phase behavior. It is an elegant route, as complicated and sensitive synthesis techniques can be avoided. Highly grafted double-comb diblock copolymers based on symmetric double hydrogen bond accepting poly(4-vinylpyridine)-block-poly(N-acryloylpiperidine) diblock copolymers and donating 3-nonadecylphenol amphiphiles are realized and studied systematically by changing the molecular weight of the copolymer. Double perpendicular lamellae-in-lamellae are formed in all complexes, independent of the copolymer molecular weight. Temperature-resolved measurements demonstrate that the supramolecular nature and ability to crystallize are responsible for the formation of such multiblock-like structures. Because of these driving forces and severe plasticization of the complexes in the liquid crystalline state, this supramolecular approach can be useful for steering self-assembly of both low- and high-molecular-weight block copolymer systems.

Surface-Induced Frustration in Solid State Polymorphic Transition of Native Cellulose Nanocrystals.

The presence of an interface generally influences crystallization of polymers from melt or from solution. Here, by contrast, we explore the effect of surface immobilization in a direct solid state polymorphic transition on individual cellulose nanocrystals (CNCs), extracted from a plant-based origin. The conversion from native cellulose I to cellulose III crystal occurred via a host-guest inclusion of ethylene diamine inside the crystal. A 60% reduction in CNC width (height) in atomic force microscopy images suggested that when immobilized on a flat modified silica surface, the stresses caused by the inclusion or the subsequent regeneration resulted in exfoliation, hypothetically, between the van der Waals bonded sheets within the crystal. Virtually no changes in dimensions were visible when the polymorphic transition was performed to nonimmobilized CNCs in bulk dispersion. With reservations and by acknowledging the obvious dissimilarities, the exfoliation of cellulose crystal sheets can be viewed as analogous to exfoliation of 2D structures like graphene from a van der Waals stacked solid. Here, the detachment is triggered by an inclusion of a guest molecule inside a host cellulose crystal and the stresses caused by the firm attachment of the CNC on a solid substrate, leading to detachment of molecular sheets or stacks of sheets.

Self-assembly of ultra-small micelles from amphiphilic lipopeptoids.

Poly(N-substituted glycine) "peptoids" constitute a promising class of peptide-mimetic materials. We introduce the self-assembly of lipopeptoids into spherical micelles ca. 5 nm in diameter as well as larger assemblies by varying the peptoid sequence design. Our results point to design rules for the self-assembly of peptoid nanostructures, enabling the creation of stable, ultra-small peptidomimetic nanospheres.

Cellulose Elementary Fibrils Assemble into Helical Bundles in S1 Layer of Spruce Tracheid Wall.

The ultrastructural organization of cellulose elementary fibrils (EFs) in wood cell wall is considered to be the prime factor regulating the material characteristics of wood in micro to macro levels and the conversion of delignified wood fibers into various products. Specifically, the complex assembly of EFs in wood cell wall limits its swellability, solubility, and reactivity, for example, in dissolution of cellulose for regeneration of textile fibers, fibril separation for the manufacture of nanocellulose, and enzymatic hydrolysis of cellulose into sugars for their subsequent fermentation to various products, like ethanol for future fossil fuels replacement. Here cryo-transmission electron tomography was applied on ultrathin spruce wood sections to reveal the EF assembly in S1 layer of the native cell wall. The resolution of these tomograms was then further enhanced by computational means. For the first time, cellulose in the intact cell wall was visualized to be assembled into helical bundles of several EFs, a structural feature that must have a significant impact on the swelling, accessibility, and solubility of woody biomass for its conversion into the aforementioned value added products.

Shear Alignment of Bola-Amphiphilic Arginine-Coated Peptide Nanotubes.

The bola-amphiphilic arginine-capped peptide RFL4RF self-assembles into nanotubes in aqueous solution. The nanostructure and rheology are probed by in situ simultaneous rheology/small-angle scattering experiments including rheo-SAXS, rheo-SANS, and rheo-GISANS (SAXS: small-angle X-ray scattering, SANS: small-angle neutron scattering, GISANS: grazing incidence small-angle neutron scattering). Nematic alignment of peptide nanotubes under shear is observed at sufficiently high shear rates under steady shear in either Couette or cone-and-plate geometry. The extent of alignment increases with shear rate. A shear plateau is observed in a flow curve measured in the Couette geometry, indicating the presence of shear banding above the shear rate at which significant orientation is observed (0.1-1 s-1). The orientation under shear is transient and is lost as soon as shear is stopped. GISANS shows that alignment at the surface of a cone-and-plate cell develops at sufficiently high shear rates, very similar to that observed in the bulk using the Couette geometry. A small isotope effect (comparing H2O/D2O solvents) is noted in the CD spectra indicating increased interpeptide hydrogen bonding in D2O, although this does not influence nanotube self-assembly. These results provide new insights into the controlled alignment of peptide nanotubes for future applications.

Structural behaviour and gene delivery in complexes formed between DNA and arginine-containing peptide amphiphiles.

We describe in depth the structure of complexes formed between DNA and two classes of arginine-containing peptide amphiphiles, namely, the lipopeptide PRW-C16 (P = proline, R = arginine, W = tryptophan, C16 = C16 : 0 alkyl chain) and the bolaamphiphile RFL4FR (R = arginine, F = phenylalanine, L = leucine). A combination of X-ray and neutron scattering provided unprecedented insights into the local structure of these complexes. Lipopeptide-based complexes self-assembled into layered structures with large-scale fractal features, hosting DNA in the interstices. Bola-amphiphile scaffolds were characterized by planar structures with DNA strands presumably sandwiched in-between peptide nanotapes. Importantly, complexation did not affect the structural integrity of DNA in either of the two complexes. The bolaamphiphile conjugates displayed high levels of molecular ordering in contrast to the liquid-crystalline features observed in lipopeptide assemblies. Peptide-DNA complexes were assessed for their potential as a means to deliver the reporter vector pEGFP-N1 into SW480 human colon carcinoma cells. Successfully transfected cells expressed green fluorescent protein. The potentiating effect of PRW-C16 on the cellular uptake of ectopic DNA was found to be much greater than that observed with RFL4FR. In contrast to the bolaamphiphile-based conjugate, the liquid-crystalline nature of the lipopeptide complex is likely to play a key role in DNA release and transfection efficiency since these weakly bound structures require lower energy expenditure during disassembly and load release.

Nanosheet Formation by an Anionic Surfactant-like Peptide and Modulation of Self-Assembly through Ionic Complexation.

The surfactant-like peptide (Ala)6-(Asp) (A6D) is shown to self-assemble into ultrathin (3 nm thick) nanosheets in aqueous solution above a critical aggregation concentration. A combination of circular dichroism and FTIR spectroscopy and X-ray diffraction shows that the nanosheets comprise interdigitated bilayers of the peptide with ß-sheet conformation. The self-assembly can be modulated by addition of hexamethylenediamine which is expected to interact with the anionic C terminus (and C-terminal D residue) of the peptide. Multiple ordered nanostructures can be accessed depending on the amount of added diamine. Nanosheet and bicontinuous network structures were observed using cryogenic-TEM and small-angle X-ray scattering. Addition of hexamethylenediamine at a sufficiently large molar ratio leads to disruption of the ordered nanostructure and the formation of a solution of A6D-diamine molecular complexes with highly charged end groups. The multiple acid-functionalized nanostructures that are accessible in this system are expected to have many applications in the fabrication of new nanomaterials.

Hierarchical Layer Engineering Using Supramolecular Double-Comb Diblock Copolymers.

The formation of unusual multilayered parallel lamellae-in-lamellae in symmetric supramolecular double-comb diblock copolymers is presented. While keeping the concentration of surfactant fixed, the number of internal layers was found to increase with molecular weight M up to 34 for the largest block copolymer. The number of internal structures n was established to scale as M0.67 and therefore enables easy design of such structures with great precision.

Stealth Amphiphiles: Self-Assembly of Polyhedral Boron Clusters.

This is the first experimental evidence that both self-assembly and surface activity are common features of all water-soluble boron cluster compounds. The solution behavior of anionic polyhedral boranes (sodium decaborate, sodium dodecaborate, and sodium mercaptododecaborate), carboranes (potassium 1-carba-dodecaborate), and metallacarboranes {sodium [cobalt bis(1,2-dicarbollide)]} was extensively studied, and it is evident that all the anionic boron clusters form multimolecular aggregates in water. However, the mechanism of aggregation is dependent on size and polarity. The series of studied clusters spans from a small hydrophilic decaborate-resembling hydrotrope to a bulky hydrophobic cobalt bis(dicarbollide) behaving like a classical surfactant. Despite their pristine structure resembling Platonic solids, the nature of anionic boron cluster compounds is inherently amphiphilic-they are stealth amphiphiles.

Self-Assembly of the Cyclic Lipopeptide Daptomycin: Spherical Micelle Formation Does Not Depend on the Presence of Calcium Chloride.

The cyclic lipopeptide Daptomycin, used as a treatment for infections where antimicrobial resistance is observed, is shown to self-assemble into spherical micelles above a critical aggregation concentration. Micelles are observed either in the absence or presence of CaCl2 , in contrast to claims in the literature that CaCl2 is required for micellization.

Self-Assembly of Telechelic Tyrosine End-Capped PEO and Poly(alanine) Polymers in Aqueous Solution.

The self-assembly in aqueous solution of three novel telechelic conjugates comprising a central hydrophilic polymer and short (trimeric or pentameric) tyrosine end-caps has been investigated. Two of the conjugates have a central poly(oxyethylene) (polyethylene oxide, PEO) central block with different molar masses. The other conjugate has a central poly(L-alanine) (PAla) sequence in a purely amino-acid based conjugate. All three conjugates self-assemble into ß-sheet based fibrillar structures, although the fibrillar morphology revealed by cryogenic-TEM is distinct for the three polymers--in particular the Tyr5-PEO6k-Tyr5 forms a population of short straight fibrils in contrast to the more diffuse fibril aggregates observed for Tyr5-PEO2k-Tyr5 and Tyr3-PAla-Tyr3. Hydrogel formation was not observed for these samples (in contrast to prior work on related systems) up to quite high concentrations, showing that it is possible to prepare solutions of peptide-polymer-peptide conjugates with hydrophobic end-caps without conformational constraints associated with hydrogelation. The Tyr5-PEO6k-Tyr5 shows significant PEO crystallization upon drying in contrast to the Tyr5-PEO2k-Tyr5 conjugate. Our findings point to the remarkable ability of short hydrophobic peptide end groups to modulate the self-assembly properties of polymers in solution in model peptide-capped "associative polymers". Retention of fluidity at high conjugate concentration may be valuable in potential future applications of these conjugates as bioresponsive or biocompatible materials, for example exploiting the enzyme-responsiveness of the tyrosine end-groups.

Self-Assembly of the Toll-Like Receptor Agonist Macrophage-Activating Lipopeptide MALP-2 and of Its Constituent Peptide.

The self-assembly of the macrophage-activating lipopeptide MALP-2 in aqueous solution has been investigated and is compared to that of the constituent peptide GNNDESNISFKEK. MALP-2 is a toll-like receptor agonist lipopeptide with diverse potential biomedical applications and its self-assembly has not previously been examined. It is found to self-assemble, above a critical aggregation concentration (cac), into remarkable "fibre raft" structures, based on lateral aggregation of ß-sheet based bilayer tapes. Peptide GNNDESNISFKEK also forms ß-sheet structures above a cac, although the morphology is distinct, comprising highly extended and twisted tape structures. A detailed insight into the molecular packing within the MALP-2 raft and GNNDESNISFKEK nanotape structures is obtained through X-ray diffraction and small-angle X-ray scattering. These results point to the significant influence of the attached lipid chains on the self-assembly motif, which lead to the raft structure for the lipopeptide assemblies.

Correction: Self-assembly of the anti-fungal polyene amphotericin B into giant helically-twisted nanotapes.

Correction for 'Self-assembly of the anti-fungal polyene amphotericin B into giant helically-twisted nanotapes' by Ian William Hamley et al., Chem. Commun., 2015, 51, 17680-17683.