Stereochemistry-Controlled Supramolecular Architectures of New Tetrahydroxy-Functionalised Amphiphilic Carbocyanine Dyes.

The syntheses of novel amphiphilic 5,5',6,6'-tetrachlorobenzimidacarbocyanine (TBC) dye derivatives with aminopropanediol head groups, which only differ in stereochemistry (chiral enantiomers, meso form and conformer), are reported. For the achiral meso form, a new synthetic route towards asymmetric cyanine dyes was established. All compounds form J aggregates in water, the optical properties of which were characterised by means of spectroscopic methods. The supramolecular structure of the aggregates is investigated by means of cryo-transmission electron microscopy, cryo-electron tomography and AFM, revealing extended sheet-like aggregates for chiral enantiomers and nanotubes for the mesomer, respectively, whereas the conformer forms predominately needle-like crystals. The experiments demonstrate that the aggregation behaviour of compounds can be controlled solely by head group stereochemistry, which in the case of enantiomers enables the formation of extended hydrogen-bond chains by the hydroxyl functionalities. In case of the achiral meso form, however, such chains turned out to be sterically excluded.

Switchable Solubility of Azobenzene-Based Bolaamphiphiles.

The ability to design amphiphiles with predictable solubility properties is of everlasting interest in supramolecular chemistry. Relevant structural parameters include the hydrophobic-hydrophilic balance and structural flexibility. In this work, we investigate the water solubility of azobenzene-based triglycerol bolaamphiphiles (TGBAs). In particular, we analyzed the structural effects of backbone hydrophobicity, flexibility, and cis/trans isomerization on the water solubility of a subset of five TGBAs. This leads to the first example of a non-ionic bolaamphiphile whose water solubility can be changed by irradiation with light. The underlying kinetics were monitored using liquid chromatography and a closer analysis of the underlying aggregation processes provides a mechanistic understanding of the light-driven dissolution process. We anticipate that the results obtained will help to engineer bolaamphiphiles with predictable solution properties in the future.

Catalytic Activity of Peptide-Nanoparticle Conjugates Regulated by a Conformational Change.

Herein, we present the design and synthesis of a catalytically active peptide-nanoparticle conjugate whose activity is regulated by a defined conformational change in the self-assembled peptide monolayer. A catalytically active peptide, designed after the heterodimeric α-helical coiled-coil principle was immobilized onto gold nanoparticles, and kinetic studies were performed according to the Michaelis-Menten model. The formed peptide monolayer at the gold nanoparticle surface accelerated p-nitrophenylacetate (pNPA) hydrolysis by 1 order of magnitude compared to the soluble peptide while exhibiting no defined secondary structure as determined by infrared (IR) and circular dichroism (CD) spectroscopy. Addition of the complementary peptide-induced coiled-coil formation while significantly hindering the pNPA hydrolysis catalyzed by the peptide-nanoparticle conjugate. The heptad repeat sequence of a coiled-coil opens up the opportunity for regulation of conformation and thus catalytic activity of peptide-nanoparticle conjugates upon interaction with a complementary coiled-coil sequence. Strategies of regulation of catalytic activity by interaction with a complementary cofactor/ligand are well-established in nature and are introduced here into rationally designed peptide-nanoparticle conjugates.

Introducing Chirality into Nonionic Dendritic Amphiphiles and Studying Their Supramolecular Assembly.

Chiral head groups have been introduced into water-soluble hydroxyl-terminated nonionic amphiphiles and the impact of the head group stereochemistry on the supramolecular ultrastructures has been studied. Enantiomeric isomers were compared with the achiral meso form and the racemic mixture by means of cryogenic transmission electron microscopy and circular dichroism spectroscopy. Structurally, all amphiphiles are composed of the first-generation hydrophilic polyglycerol head group coupled to a single hydrophobic hexadecyl chain through an amide linkage and diaromatic spacer. The enantiomers aggregate to form twisted ribbons with uniform handedness, whereas the meso stereoisomer and racemic mixture produce elongated assemblies, namely, tubules and platelets, but without a chiral ultrastructure. Simulations on the molecular packing geometries of the stereoisomers indicate different preferential assembly routes that explain the individual supramolecular aggregation behavior.

Amphiphilic perylene-calix[4]arene hybrids: synthesis and tunable self-assembly.

The first highly water-soluble perylene-calix[4]arene hybrid with the calixarene scaffold acting as a structure-determining central platform is presented. In this tetrahedrally shaped amphiphilic architecture the hydrophilic and hydrophobic subunits are oriented at the opposite side of the calixarene platform. The hydrophobic part contains the two perylene diimide moieties, which enable strong π-π interactions in self-assembly processes. Two hydrophilic Newkome-type dendrons provide sufficient water solubility at slightly basic conditions. The tetrahedrally shaped amphiphile displays an unprecedented aggregation behavior down to concentrations as low as 10(-7) mol L(-1). The intriguing self-assembly process of the compound in water as well as under changed polarity conditions, achieved by addition of THF, could be monitored by the complemented use of cryogenic transmission electron microscopy (cryo-TEM), UV-vis spectroscopy, and fluorescence spectroscopy. Molecular-dynamics and molecular modeling simulations helped in understanding the interplay of supramolecular and optical behavior.

Impact of multivalent charge presentation on peptide-nanoparticle aggregation.

Strategies to achieve controlled nanoparticle aggregation have gained much interest, due to the versatility of such systems and their applications in materials science and medicine. In this article we demonstrate that coiled-coil peptide-induced aggregation based on electrostatic interactions is highly sensitive to the length of the peptide as well as the number of presented charges. The quaternary structure of the peptide was found to play an important role in aggregation kinetics. Furthermore, we show that the presence of peptide fibers leads to well-defined nanoparticle assembly on the surface of these macrostructures.

Hierarchically ordered self-assembly of amphiphilic bifullerenes.

A series of novel functionalised dumbbell-shaped bifullerenes in which two [5.0] pentakis-adducts of C60 are covalently connected by cyclic bismalonates were synthesised. These dimeric compounds, carrying various combinations of hydrophilic and hydrophobic addends, self-assemble in aqueous solution towards supramolecular architectures of different structural complexity as observed by cryogenic transmission electron microscopy (cryo-TEM). The detailed analysis of the image data revealed an unprecedented hierarchical aggregation behaviour. Whereas completely hydrophilic substituted bifullerenes formed profoundly monodisperse populations of small oligomeric elementary micelles consisting of only three or four bifullerene molecules in a supposedly bent conformation, their amphiphilic equivalents underwent a hierarchical two-step assembly process towards larger spherical and even rod-like structures. The data suggest that the hierarchical assembly process is driven by hydrophobic interactions of preformed tetrameric elementary micelles.

Non-ionic dendronized multiamphiphilic polymers as nanocarriers for biomedical applications.

A new class of non-ionic dendronized multiamphiphilic polymers is prepared from a biodegradable (AB)n-type diblock polymer synthesized from 2-azido-1,3-propanediol (azido glycerol) and polyethylene glycol (PEG)-600 diethylester using Novozym-435 (Candida antarctica lipase) as a biocatalyst, following a well-established biocatalytic route. These polymers are functionalized with dendritic polyglycerols (G1 and G2) and octadecyl chains in different functionalization levels via click chemistry to generate dendronized multiamphiphilic polymers. Surface tension measurements and dynamic light scattering studies reveal that all of the multiamphiphilic polymers spontaneously self-assemble in aqueous solution. Cryogenic transmission electron microscopy further proves the formation of multiamphiphiles towards monodisperse spherical micelles of about 7-9 nm in diameter. The evidence from UV-vis and fluorescence spectroscopy suggests the effective solubilization of hydrophobic guests like pyrene and 1-anilinonaphthalene-8-sulfonic acid within the hydrophobic core of the micelles. These results demonstrate the potential of these dendronized multiamphiphilic polymers for the development of prospective drug delivery systems for the solubilization of poorly water soluble drugs.

Fluorinated dendritic amphiphiles, their stomatosome aggregates and application in enzyme encapsulation.

Enzymes are more selective and efficient than synthetic catalysts but are limited by difficult recycling. This is overcome by immobilisation, namely through encapsulation, with the main drawback of this method being slow diffusion of products and reactants, resulting in effectively lowered enzyme activity. Fluorinated dendritic amphiphiles were reported to self-assemble into regularly perforated bilayer vesicles, so-called "stomatosomes". It was proposed that they could be promising novel reaction vessels due to their increased porosity while retaining larger biomolecules at the same time. Amphiphiles were synthesised and their aggregation was analysed by cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) in buffered conditions necessary for enzyme encapsulation. Urease and albumin were encapsulated using the thin-film hydration method and investigated by confocal and time-gated stimulated emission depletion microscopy (gSTED). Their release was then used to probe the selective retention of cargo by stomatosomes. Free and encapsulated enzyme activity were compared and their capacity to be reused was evaluated using the Berthelot method. Urease was successfully encapsulated, did not leak out at room temperature, and showed better activity in perforated vesicles than in closed vesicles without perforations. Encapsulated enzyme could be reused with retained activity over 8 cycles using centrifugation, while free enzyme had to be filtrated. These results show that stomatosomes may be used in enzyme immobilisation applications and present advantages over closed vesicles or free enzyme.

Synthesis and Linker-Controlled Self-Assembly of Dendritic Amphiphiles with Branched Fluorinated Tails.

Amphiphiles containing fluorinated segments tend to aggregate in the aqueous solution into structure of lower curvature than their hydrocarbon analogs due to their larger diameter. A benefit of supramolecular structures incorporating fluorine moieties is their high electron density, which can be viewed in cryo-TEM with better contrast than their hydrogenated forms. A modular approach has been developed for the synthesis of a new family of nonionic branched amphiphiles consisting of oligoglycerol units (G2) as the hydrophilic part and a branched fluorinated (F27) hydrophobic part. The design of this hydrophobic moiety allows to achieve a higher fluorine density than the previously used straight-chain perfluoroalkanes. Two different chemical approaches, amide, and triazole, are used to link the hydrophilic and hydrophobic segments. In addition, the aggregation behavior is investigated by dynamic light scattering (DLS) and cryo-TEM. The measurements prove the formation of multivesicular (MVVs) and multilamellar (MLVs) vesicles as well as smaller unilamellar vesicles. Further, the cell viability test proves the low cell toxicity of these nanoarchitectures for potential biomedical applications.

Rational design of amphiphilic fluorinated peptides: evaluation of self-assembly properties and hydrogel formation.

Advanced peptide-based nanomaterials composed of self-assembling peptides (SAPs) are of emerging interest in pharmaceutical and biomedical applications. The introduction of fluorine into peptides, in fact, offers unique opportunities to tune their biophysical properties and intermolecular interactions. In particular, the degree of fluorination plays a crucial role in peptide engineering as it can be used to control the characteristics of fluorine-specific interactions and, thus, peptide conformation and self-assembly. Here, we designed and explored a series of amphipathic peptides by incorporating the fluorinated amino acids (2S)-4-monofluoroethylglycine (MfeGly), (2S)-4,4-difluoroethylglycine (DfeGly) and (2S)-4,4,4-trifluoroethylglycine (TfeGly) as hydrophobic components. This approach enabled studying the impact of fluorination on secondary structure formation and peptide self-assembly on a systematic basis. We show that the interplay between polarity and hydrophobicity, both induced differentially by varying degrees of side chain fluorination, does affect peptide folding significantly. A greater degree of fluorination promotes peptide fibrillation and subsequent formation of physical hydrogels in physiological conditions. Molecular simulations revealed the key role played by electrostatically driven intra-chain and inter-chain contact pairs that are modulated by side chain fluorination and give insights into the different self-organization behaviour of selected peptides. Our study provides a systematic report about the distinct features of fluorinated oligomeric peptides with potential applications as peptide-based biomaterials.

Aggregation of Amphiphilic Carbocyanines: Fluorination Favors Cylindrical Micelles over Bilayered Tubes.

The synthesis of a new amphiphilic 5,5',6,6'-tetrachlorobenzimidacarbocyanine dye derivative with -(CH2)2-(CF2)5-CF3 chains attached to the nitrogen atoms in the 1,1'-position, CF8O3, is reported. Depending on the dye concentration and the addition of MeOH, CF8O3 forms J- and H-aggregates in aqueous solutions. The aggregation behavior was investigated using steady-state absorption, linear dichroism, and fluorescence spectroscopy, as well as by cryogenic transmission electron microscopy (cryo-TEM). The J-band of the MeOH-free solution is monomer-like, rather broad, and less red-shifted with respect to the monomer absorption, indicating weak excitonic coupling and disorder effects. Cryo-TEM reveals a diversity of supramolecular structures, wherein linear and branched cylindrical micelles dominate. It is concluded that the high stiffness of fluoroalkyl chains does not allow the chains to splay and completely fill up the hydrophobic gap between opposing chromophores. This destabilizes the bilayers and favors the micellar structure motifs instead. The aggregates appearing at 30% MeOH show a split absorption spectrum consisting of a broad blue-shifted H-band and an accompanying sharp red-shifted J-band with perpendicular polarizations. These HJ-type aggregates are also composed of micellar fibers, but these bundle into rope-like strands. For 10% MeOH, a narrow bilayered tube is the dominating morphology. The observed MeOH dependence of aggregation reveals a clear cosolvent effect.

Sodium effect on self-organization of amphiphilic carboxylates: formation of structured micelles and superlattices.

Not only the self-aggregation of dendritic polycarboxylates into structurally persistent micelles, but also that of the micelles themselves into superlattices is controlled by alkali-metal counterions and shows a pronounced sodium effect. Our combined experimental and computational work has revealed the formation of superlattices for the first time. The behavior of a variety of amphiphilic carboxylates and the different effects of the alkali cations Li(+), Na(+), and K(+) have been investigated by conductivity measurements, cryogenic transmission electron microscopy (cryo-TEM), and molecular-dynamics (MD) simulations. Together, these show that sodium salts of the amphiphiles give the most stable micelles, followed by lithium and potassium. Our results suggest that ion multiplets in bridging positions, rather than contact ion pairs, are responsible for the enhanced stability and the formation of hexagonally ordered superlattices with sodium counterions. Potassium ions do not form such ion multiplets and cannot therefore induce aggregation of the micelles. This sodium effect has far-reaching consequences for a large number of biological and technical systems and sheds new light on the origin of specific-ion effects.

Self-assembly of structurally persistent micelles is controlled by specific-ion effects and hydrophobic guests.

A combined study using cryo-TEM experiments and molecular dynamics simulations reveals remarkable details of the factors that affect the self-organization of specifically designed T-shaped amphiphilic dendrimers upon treatment of an aqueous solution with ultrasound under a layer of hexane. This treatment leads to dodecameric, structured micelles rather than the heptameric ones observed without hexane. Three-dimensional reconstruction of the cryo-TEM images provides very detailed structures of the micelles, and molecular dynamics simulations suggest that approximately 36 hexane molecules are needed to stabilize the dodecameric micelles. Sodium counterions are found to exert a significant stabilizing effect that results in an apparent attraction between the highly negatively charged polycarboxylate headgroups. DFT calculations support the observation that the formation of ion multiplets is especially crucial for this stabilizing counterion effect, which reduces headgroup repulsion. This and the increased hydrophobic stabilization that results from the hexane-enlarged core of the micelle lead to stable dodecameric micelles. The specific effects found for sodium counterions are largely absent for potassium.

Non-ionic PEG-oligoglycerol dendron conjugated nano-carriers for dermal drug delivery.

A new class of non-ionic amphiphiles have been synthesised using a combination of polyethylene glycol (PEG) and oligoglycerol dendrons as hydrophilic units and an alkoxy aryl moiety as hydrophobic unit. The resulting amphiphiles were found to aggregate in aqueous medium. Their aggregation behaviour was studied using dynamic light scattering (DLS), fluorescence spectroscopy, and cryogenic electron microscopy (cryo-TEM). The inner hydrophobic core of these aggregates in aqueous medium is capable of encapsulating lipophilic guest molecules. The encapsulation behaviour was studied using Nile red as a hydrophobic dye as well as Curcumin and Dexamethasone as hydrophobic drug candidates. Furthermore, for biological evaluation, cytotoxicity and cellular uptake was studied using different cancer cell lines. The biomedical application of synthesised amphiphiles was further investigated for dermal drug delivery on excised human skin using Nile red encapsulated in the nanocarrier. The release profile of drug/dye encapsulated amphiphiles was studied under physiochemical conditions in the presence of immobilized lipase Novozym 435.

Electron cryomicroscopy reveals different F1+F2 protein States in intact parainfluenza virions.

Electron cryomicrographs of intact parainfluenza virus 5 (PIV5) virions revealed two different surface structures, namely, a continuous layer and distinct individual spikes. The structure of these spikes reconstructed from intact virions was compared with known F ectodomain structures and was found to be different from the prefusion PIV5 F0 structure but, surprisingly, very similar to the human PIV3 F postfusion structure. Hence, we conclude that the individual F1+F2 spikes in intact PIV5 virions also correspond to the postfusion state. Since the observed fusion activity of PIV5 virions has to be associated with prefusion F1+F2 proteins, they have necessarily to be localized in the continuous surface structure. The data therefore strongly suggest that the prefusion state of the F1+F2 protein requires stabilization, most probably by the association with hemagglutinin-neuraminidase. The conversion of F1+F2 proteins from the prefusion toward the postfusion state while embedded in the virus membrane is topologically difficult to comprehend on the basis of established models and demands reconsideration of our current understanding.

Supramolecular assembly of self-labeled amphicalixarenes.

The synthesis and precise supramolecular organization of new amphicalixarene 4 bearing four rodlike aligned fluorescent terephthalic benzamide moieties in the upper rim is reported. The aggregation of 4 was monitored by a combination of fluorescence, conductometry, and cryo-TEM measurements at different pH values and revealed significant structural differences. Most interestingly, we found that exactly 12 molecules of 4 assemble to form a spherical and structurally persistent micelle at pH 7, which coexists with rodlike micelles. In contrast to other examples of structurally defined micelles reported previously, this new type of self-labeled amphiphile serves as a fluorescence reporter with the terephthalic units additionally providing an extended cavity structure in the hydrophilic part, which facilitates the inclusion of guest molecules. In addition to the observed formation of well-defined micelles, the other highly important finding from this study is the fact that guest molecules directly influence the micellar organization because they can interact with both the free amphiphiles below the critical micelle concentration (cmc) or the micellar aggregate itself. These two types of interactions are especially pronounced in the case of the cationic pyrene derivative 10, which binds electrostatically with the amphiphile 4. In addition, a very unique membrane structure exhibiting a regular hexagonal pattern of 5 nm pores is formed by 4 at pH 4.

Counterions control the self-assembly of structurally persistent micelles: theoretical prediction and experimental observation of stabilization by sodium ions.

We show by molecular-dynamics (MD) simulations and cryo-transmission electron microscopy (cryo-TEM) experiments that the size and form of structurally persistent micelles formed by the T-shaped amphiphile 1 are controlled by the counterions. The two techniques reveal that the micelles are specifically stabilized by sodium counterions relative to potassium ions. Both the simulations and the cryo-TEM experiments suggest that the micelles are stabilized by strongly conserved hydrated contact ion pairs with sodium counterions but not with potassium ions. We suggest that the TEM is observing local high density due to hydrated carboxylate/sodium ion pairs at the surface of the micelle. A high concentration of such structures is found in MD simulations with sodium counterions, but not with potassium.

Nonionic Dendritic and Carbohydrate Based Amphiphiles: Self-Assembly and Transport Behavior.

Herein, a new series of non-ionic dendritic and carbohydrate based amphiphiles is synthesized employing biocompatible starting materials and studied for supramolecular aggregate formation in aqueous solution. The dendritic amphiphiles 12 and 13 possessing poly(glycerol) [G2.0] as hydrophilic unit and C-10 and C-18 hydrophobic alkyl chains, respectively, exhibit low critical aggregation concentration (CAC) in the order of 10-5 m and hydrodynamic diameters in the 8-10 nm range and supplemented by cryogenic transmission electron microscopy. Ultraviolet-visible (UV-Vis) and fluorescence spectroscopy suggests the effective solubilization of hydrophobic guests by the self-assembled architectures, with the nanotransporters 12 and 13 possessing the highest encapsulation efficiency of 80.74 and 98.03% for curcumin. Efficient uptake of encapsulated curcumin in adenocarcinomic human alveolar basal epithelial (A549) cells is observed by confocal laser scanning microscopy. Amphiphiles 12 and 13 are non-cytotoxic at the concentrations studied, however, curcumin encapsulated samples efficiently reduce the viability of A549 cells in vitro. Experimental studies indicate the ability of amphiphile 13 to encapsulate 1-anilinonaphthalene-8-sulfonic acid (ANS) and curcumin with binding constant of 1.16 × 1055 m-1 and 1.43 × 106 m-1 , respectively. Overall, our findings demonstrate the potential of these dendritic amphiphiles for the development of prospective nanocarriers for the solubilization of hydrophobic drugs.

Synthesis of non-ionic bolaamphiphiles and study of their self-assembly and transport behaviour for drug delivery applications.

A series of four bolaamphiphiles with different hydrophilic units has been synthesised. All the amphiphiles were well characterised from their physiochemical data. The aggregation tendency of newly synthesised amphiphiles was studied using fluorescence spectroscopy, dynamic light scattering (DLS), and cryogenic electron microscopy (cryo-TEM). Furthermore, their application as nanocarriers for hydrophobic guests was demonstrated by using two established standards, i.e. the dye Nile red and the drug nimodipine. A cytotoxicity and cellular uptake study has been carried out using A549 cells. Due to the presence of an ester linkage in PEG based bolaamphiphiles, a drug release study was performed in the presence of an immobilized enzyme Novozym-435 (a lipase).