Dynamic octopus amphiphiles as powerful activators of DNA transporters: differential fragrance sensing and beyond

We report the design, synthesis and evaluation of dynamic "octopus" amphiphiles with emphasis on their efficiency as activators in synthetic membrane-based sensing systems. Previously, we found that the in situ treatment of charged hydrazides with hydrophobic aldehydes or ketones gives amphiphilic counterion activators of polyion transporters in lipid bilayers, and that their efficiency increases with the number of their hydrophobic tails. Herein, we expand this series to amphiphiles with one cationic head (guanidinium or ammonium) and four exchangeable hydrophobic tails. These results, with the highest number of tails reported to date, confirm that dynamic octopus amphiphiles provide access to maximal activity and selectivity. Odorants, such as muscone, carvone, or anisaldehyde are used to outline their usefulness in differential sensing systems that operate based on counterion-activated DNA transporters in fluorogenic vesicles. The enhanced ability of octopus amphiphiles to enable the discrimination of enantiomers as well as that of otherwise intractable ortho, meta, and para isomers and short cyclo-/alkyl tails is demonstrated. These findings identify dynamic octopus amphiphiles as being promising for application to differential sensing, "fragrant" cellular uptake, and slow release.     

Synthesis of an enlarged library of dynamic DNA activators with oxime, disulfide and hydrazone bridges

Dynamic amphiphiles have a "bridge" between their charged head and their hydrophobic tails. The presence of dynamic covalent bonds is of interest for differential and biosensing applications as well as for rapid access to the libraries needed to screen for gene delivery or cellular uptake of siRNA. However, efforts to develop libraries have so far concentrated on hydrazone bridges to monocationic heads. Here, we report synthesis efforts to enlarge this focused library with oxime and disulfide bridges and dynamic amphiphiles with more than one positive charge. Evaluation in fluorogenic vesicles reveals best activation of DNA as ion transporters by dynamic amphiphiles with dendritic scaffolds, doubly charged heads and four tails. Moreover, oximes, contrary to hydrazones, remain active under acidic conditions. Linear elongation of dendritic head-groups seems to cause increasing detergent effects and should therefore be avoided.     

Amphiphilic dynamic NDI and PDI probes: imaging microdomains in giant unilamellar vesicles

Dynamic amphiphiles provide access to transmembrane ion transport, differential sensing and cellular uptake. In this report, we introduce dynamic amphiphiles with fluorescent tails. Core-substituted naphthalenediimides (cNDIs) and perylenediimides (cPDIs) are tested. Whereas the latter suffer from poor partitioning, dynamic cNDI amphiphiles are found to be purifiable by RP-HPLC, to partition selectively into liquid-disordered (Ld) microdomains of mixed lipid bilayers and to activate DNA as transporters. Importantly, fluorescence properties, partitioning and activity can be modulated by changes in the structure of mixed amphiphiles. These results confirm the potential of dynamic fluorescent amphiphiles to selectively label extra- and intracellular membrane domains and visualize biological function.     

Specific binding structures of dendrimers on lipid bilayer membranes

Dissipative particle dynamics simulations are used to study the specific binding structures of polyamidoamine (PAMAM) dendrimers on amphiphilic membranes and the permeation mechanisms. Mutually consistent coarse-grained (CG) models both for PAMAM dendrimers and for dimyristoylphosphatidylcholine (DMPC) lipid molecules are constructed. The PAMAM CG model describes correctly the conformational behavior of the dendrimers, and the DMPC CG model can properly give the surface tension of the amphiphilic membrane. A series of systematic simulations is performed to investigate the binding structures of the dendrimers on membranes with varied length of the hydrophobic tails of amphiphiles. The permeability of dendrimers across membranes is enhanced upon increasing the dendrimer size (generation). The length of the hydrophobic tails of amphiphiles in turn affects the dendrimer conformation, as well as the binding structure of the dendrimer-membrane complexes. The negative curvature of the membrane formed in the dendrimer-membrane complexes is related to dendrimer concentration. Higher dendrimer concentration together with increased dendrimer generation is observed to enhance the permeability of dendrimers across the amphiphilic membranes.     

Sunfish cationic amphiphiles: toward an adaptative lipoplex morphology

A detailed physicochemical study is presented on a new class of cationic amphiphiles, Sunfish amphiphiles, recently designed, synthesized, and tested for gene delivery. These materials have two hydrophobic tails, connected to the cationic pyridinium headgroup at the 1- and 4-positions. Two extreme morphologies can be visualized, i.e. one by back-folding involving association of both tails at one side of the pyridinium ring and one by independent unfolding of the tails, the two molecular geometries leading to considerable differences in the aggregate morphology. The behavior of six members of the Sunfish family in mixtures with DOPE, applying different conditions relevant for transfection, has been studied by a combination of techniques (DLS, DSC, NMR, SAXS, Cryo-TEM, fluorescence, etc.). The effects of structural parameters such as the presence of unsaturation in the tails and length of the alkyl chains on the properties of the aggregates have been assessed. A correlation of these structural data with cellular transfection efficiencies reveals that the highest transfection efficiency is obtained with those amphiphiles that are easily hydrated, form fluid aggregates, and undergo a transition to the inverted hexagonal phase in the presence of plasmid DNA (p-DNA) at physiological ionic strength.     

Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery

A number of prior studies have demonstrated that the DNA-binding and gene transfection efficacies of cationic amphiphiles crucially depend on their various structural parameters including hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar headgroups connected by the same ester linker group. The only structural difference between the cationic amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2 cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene delivery efficacies of cationic liposomes.     

Spider-web amphiphiles as artificial lipid clusters: design, synthesis, and accommodation of lipid components at the air-water interface

As a novel category of two-dimensional lipid clusters, dendrimers having an amphiphilic structure in every unit were synthesized and labeled "spider-web amphiphiles". Amphiphilic units based on a Lys-Lys-Glu tripeptide with hydrophobic tails at the C-terminal and a polar head at the N-terminal are dendrically connected through stepwise peptide coupling. This structural design allowed us to separately introduce the polar head and hydrophobic tails. Accordingly, we demonstrated the synthesis of the spider-web amphiphile series in three combinations: acetyl head/C16 chain, acetyl head/C18 chain, and ammonium head/C16 chain. All the spider-web amphiphiles were synthesized in satisfactory yields, and characterized by 1H NMR, MALDI-TOFMS, GPC, and elemental analyses. Surface pressure (pi)-molecular area (A) isotherms showed the formation of expanded monolayers except for the C18-chain amphiphile at 10 degrees C, for which the molecular area in the condensed phase is consistent with the cross-sectional area assigned for all the alkyl chains. In all the spider-web amphiphiles, the molecular areas at a given pressure in the expanded phase increased in proportion to the number of units, indicating that alkyl chains freely fill the inner space of the dendritic core. The mixing of octadecanoic acid with the spider-web amphiphiles at the air-water interface induced condensation of the molecular area. From the molecular area analysis, the inclusion of the octadecanoic acid bears a stoichiometric characteristic; i.e., the number of captured octadecanoic acids in the spider-web amphiphile roughly agrees with the number of branching points in the spider-web amphiphile.     

Bola-Amphiphilic Glycodendrimers: New Carbohydrate-Mimicking Scaffolds to Target Carbohydrate-Binding Proteins

Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.     

Self-assembly, DNA complexation, and pH response of amphiphilic dendrimers for gene transfection

Cationic lipids and polymers are routinely used for cell transfection, and a variety of structure-activity relation data have been collected. Few studies, however, focus on the structural aspects of self-assembly as a crucial control parameter for gene delivery. We present here the observations collected for a set of cationic dendritic amphiphiles based on a stiff tolane core (1-4) that are built from identical subunits but differ in the number and balance of their hydrophobic and cationic hydrophilic moieties. We established elsewhere that vectors 3 and 4 have promising transfection properties. Scanning probe microscopy (AFM, STM), cryo-transmission electron microscopy (cryo-TEM), and Langmuir techniques provide insight into the self-assembly properties of the molecules under physiological conditions. Furthermore, we present DNA and pH "jump" experiments where we study the response of Langmuir films to a sudden increase in DNA concentration or a drop in pH. We find that the primary self-assembly of the amphiphile is of paramount importance and influences DNA binding, serum sensitivity, and pH response of the vector system.     

In situ hydrophobized, shape-anisotropic nanoparticles for composite materials

An inverse emulsion technique which allows the anisotropic growth of a broad variety of inorganic nanoparticles, together with an efficient hydrophobization, is described. This method is based upon the combined use of amphiphilic copolymers, which act as emulsifiers as well as compatibilizers, and structure-directing agents that control the crystallization of the inorganic nanoparticles. As a consequence, water-soluble, structure-directing agents can now be applied for the synthesis of hydrophobic, shape-anisotropic nanocrystals. More precisely, spherical, rod-like, and branched CdS as well as Au nanoparticles were prepared. Due to their excellent hydrophobization, these particles were homogeneously incorporated into a poly(2-ethylhexyl methacrylate) matrix. Their shape-dependent properties were transferred to nanocomposites as demonstrated for branched CdS nanocrystals. In comparison to more traditional materials composed of branched CdS nanoparticles, which are stabilized by low molecular weight amphiphiles, our composites show much less scattering. This is due to the homogenous distribution of the nanoparticles in the matrix.     

Air–water interfacial behavior of amphiphilic peptide analogs of synthetic chloride ion transporters

A family of heptapeptide-based chloride transporters (called synthetic anion transporters, SATs) were designed to insert into phospholipid membrane bilayers and form pores. Many of these compounds have proved to be chloride selective transporters. The transporters were designed to incorporate hydrophilic heptapeptides that could serves as headgroups and hydrocarbon tails that could serve as hydrophobic membrane anchors. Insertion of the SAT molecules into a bilayer requires approach to and insertion at the aqueous-membrane surface. The studies reported here were conducted to model and understand this process by studying SAT behavior at the air–water interface. A Langmuir trough was used to obtain surface pressure–area isotherm data. These data for amphiphilic SATs were augmented by Brewster angle microscopy (BAM), molecular modeling, and calculations of the hydrophobicity parameter log P. The analyses showed that the heptapeptide (hydrophilic) module of the SAT molecule rested on the water surface while the dialkyl (hydrophobic) tails oriented themselves in the air, perpendicular to the water surface. Brewster angle microscopy visually confirmed a high order of molecular organization. Results from these studies are consistent with the previously proposed mechanism of SAT membrane insertion and pore formation.     

Toroid morphology by ABC-type amphiphilic rod-coil molecules at the air-water interface

The interfacial and aggregation behavior of the ABC-type amphiphilic molecules with semirigid dumbbell-shaped core and variable length of hydrophobic branched tails (R=(CH2)nCH3 with n=5 (1), 9 (2), 13 (3)) were investigated. At low surface pressure, smooth, uniform monolayers were formed at the air-water interface by molecules 1 and 2, whereas for molecule 3 unique 2D toroid aggregates have been formed. These aggregates were relatively stable within a range of surface pressure and spreading solution concentration. Upon compression, the 2D toroid aggregates collapsed into large, round 3D aggregates. Finally, the choice of spreading solvent has a great influence on aggregation formation into 2D or 3D micelles as a result of the variable balance of the hydrophobic interactions of branched tails and the pi-pi stacking interaction between aromatic segments.     

The High‐Throughput Synthesis and Phase Characterisation of Amphiphiles: A Sweet Case Study

A new method for the discovery of amphiphiles by using high‐throughput (HT) methods to synthesise and characterise a library of galactose‐ and glucose‐containing amphiphilic compounds is presented. The copper‐catalysed azide–alkyne cycloaddition (CuAAC) “click” reaction between azide‐tethered simple sugars and alkyne‐substituted hydrophobic tails was employed to synthesise a library of compounds with systematic variations in chain length and unsaturation in a 24‐vial array format. The liquid–crystalline phase behaviour was characterised in a HT manner by using synchrotron small‐angle X‐ray scattering (SSAXS). The observed structural variation with respect to chain parameters, including chain length and degree of unsaturation, is discussed, as well as hydration effects and degree of hydrogen bonding between head groups. The validity of our HT screening approach was verified by resynthesising a short‐chain glucose amphiphile. A separate phase analysis of this compound confirmed the presence of numerous lyotropic liquid–crystalline phases.     

Synthesis,physicochemical characterization,and self‐assembly of linear,dibranched, and miktoarm semifluorinated triphilic polymers

Linear, dibranched, and miktoarm amphiphiles containing both hydrophobic and fluorophilic moieties were synthesized and characterized in an attempt to elucidate the relationship between semifluorinated amphiphile structure and aggregate behavior in aqueous solution. For the linear and dibranched amphiphiles, there was an exponential decrease in critical aggregation concentration (CMC) and a logarithmic increase in core microviscosity with increasing length of the fluorocarbon segments; while the miktoarm architecture produced no notable trend in microviscosity or CMC. Furthermore, the linear and dibranched surfactants showed enhanced kinetic stability, dissociating more slowly in the presence of human serum than did either the dibranched or miktoarm amphiphiles. Finally, encapsulation studies with the hydrophobic drug paclitaxel (PTX) showed that the ability to solubilize and retain PTX increased with the presence and with the increasing size of the fluorocarbon moiety for both the linear and dibranched amphiphiles, while no such trend was observed for the miktoarm amphiphiles. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 3324–3336     

Unraveling the mechanism of nanotube formation by chiral self-assembly of amphiphiles

The self-assembly of nanotubes from chiral amphiphiles and peptide mimics is still poorly understood. Here, we present the first complete path to nanotubes by chiral self-assembly studied with C(12)-β(12) (N-α-lauryl-lysyl-aminolauryl-lysyl-amide), a molecule designed to have unique hybrid architecture. Using the technique of direct-imaging cryo-transmission electron microscopy (cryo-TEM), we show the time-evolution from micelles of C(12)-β(12) to closed nanotubes, passing through several types of one-dimensional (1-D) intermediates such as elongated fibrils, twisted ribbons, and coiled helical ribbons. Scattering and diffraction techniques confirm that the fundamental unit is a monolayer lamella of C(12)-β(12), with the hydrophobic tails in the gel state and β-sheet arrangement. The lamellae are held together by a combination of hydrophobic interactions, and two sets of hydrogen-bonding networks, supporting C(12)-β(12) monomers assembly into fibrils and associating fibrils into ribbons. We further show that neither the "growing width" model nor the "closing pitch" model accurately describe the process of nanotube formation, and both ribbon width and pitch grow with maturation. Additionally, our data exclusively indicate that twisted ribbons are the precursors for coiled ribbons, and the latter structures give rise to nanotubes, and we show chirality is a key requirement for nanotube formation.     

β-Cyclodextrin-based polycationic amphiphilic "click" clusters: effect of structural modifications in their DNA complexing and delivery properties

Monodisperse facial amphiphiles consisting of a β-cyclodextrin (βCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hydrophobic chains at the secondary oxygens have been prepared by implementing two very robust "click" methodologies, namely cuprous cation-catalyzed azide-alkyne cycloaddition (CuAAC) and thiourea-forming reaction. Most interestingly, the use of solid-supported Cu(I) catalysts was found to be very well suited for multiple CuAAC while facilitating purification of the C(7)-symmetric macromolecular triazole adducts. The strategy is compatible with molecular diversity-oriented approaches, which has been exploited to generate a small library of click polycationic amphiphilic CDs (paCDs) for assessing the influence of structural modifications in the ability to complex, compact, and protect pDNA and the efficiency of the resulting paCD:pDNA nanocomplexes (CDplexes) to deliver DNA into cells and promote transfection. The results indicate that fine-tuning the hydrophilic/hydrophobic balance is critical to achieve optimal self-assembling properties and stability of the resulting CDplexes in saline- and serum-containing media. Triazole-type paCDs were, in general, less efficient in promoting gene transfection than thiourea-type derivatives. Nevertheless, the current body of results support that the "dual click" approach implying sequential CuAAC and thiourea-forming reactions represents a versatile strategy to optimize the gene delivery capabilities of cyclodextrin-based facial amphiphiles.     

Phase behavior of amphiphiles at liquid crystals/water interface: A coarse-grained molecular dynamics study

We present a coarse-grained molecular dynamics simulation study of phase behavior of amphiphilic monolayers at the liquid crystal （LC）/water interface. The results revealed that LCs at interface can influence the lateral ordering of amphiphiles. Particularly, the amphiphile tails along with perpendicularly penetrated LCs between tails undergo a two-dimension phase transition from liquid-expanded into a liquid-condensed phase as their area density at interface reaches 0.93. While, the liquid-condensed phase of the monolayer never appears at oil/water interface with isotropic shape oil particles. These findings reveal the penetration of anisotropic LC can promote ordered lateral organization of amphiphiles. Moreover, we find the phase transition point is shifted to lower surface coverage of amphiphiles when the LCs have larger affinity to the amphiphile tails.     

Novel glucose derived non-ionic gemini surfactants as reverse micellar systems for encapsulation of d- and l-enantiomers of some aromatic α-amino acids in n-hexane

Novel glucose-based non-ionic gemini amphiphiles comprising two sugar head groups, two hydrophobic tails having chain length of C₁₂, C₁₄, and C₁₈ and a –CH₂–Ar–CH₂– spacer have been synthesized. The head groups of the geminis consist of glucose entities (with reducing function blocked in cyclic acetal group) connected through C-6 to tertiary amines. These amphiphiles were explored as reverse micellar systems, for the encapsulation of d- and l-enantiomers of ultraviolet-absorbing aromatic α-amino acids histidine (H), phenylalanine (F), tyrosine (Y) and tryptophan (W) in n-hexane, without any added water. Reverse micellar studies revealed that aromatic α-amino acids were encapsulated in the sequence H?>?F?>?Y?>?W. In most cases, specifically for F, d-enantiomer was found better encapsulated than l-enantiomer in the reverse micellar probes of the gemini surfactants.     

Chelating DTPA amphiphiles: ion-tunable self-assembly structures and gadolinium complexes

A series of chelating amphiphiles and their gadolinium (Gd(iii)) metal complexes have been synthesized and studied with respect to their neat and lyotropic liquid crystalline phase behavior. These amphiphiles have the ability to form ion-tunable self-assembly nanostructures and their associated Gd(III) complexes have potential as magnetic resonance imaging (MRI) contrast enhancement agents. The amphiphiles are composed of diethylenetriaminepentaacetic acid (DTPA) chelates conjugated to one or two oleyl chain(s) (DTPA-MO and DTPA-BO), or isoprenoid-type chain(s) of phytanyl (DTPA-MP and DTPA-BP). The thermal phase behavior of the neat amphiphiles was examined by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and cross polarizing optical microscopy (POM). Self-assembly of neat amphiphiles and their associated Gd complexes, as well as their lyotropic phase behavior in water and sodium acetate solutions of different ionic strengths, were examined by POM and small and wide angle X-ray scattering (SWAXS). All neat amphiphiles exhibited lamellar structures. The non-complexed amphiphiles showed a variety of lyotropic phases depending on the number and nature of the hydrophobic chain in addition to the ionic state of the hydration. Upon hydration with increased Na-acetate concentration and the subtle changes in the effective headgroup size, the interfacial curvature of the amphiphile increased, altering the lyotropic liquid crystalline structures towards higher order mesophases such as the gyroid (Ia3d) bicontinuous cubic phase. The chelation of Gd with the DTPA amphiphiles resulted in lamellar crystalline structures for all the neat amphiphiles. Upon hydration with water, the Gd-complexed mono-conjugates formed micellar or vesicular self-assemblies, whilst the bis-conjugates transformed only partially into lyotropic liquid crystalline mesophases.