SANS contrast matching for the unambiguous localization of anionic dye in cationic surfactant micelles.

Contrast variation in small-angle neutron scattering (SANS) was successfully applied to localize the anionic azo dye Blue in co-assemblies with the cationic surfactant dodecyltrimethylammoniumbromide (DTAB). For this purpose, the scattering contrast between DTAB and the aqueous solvent was eliminated by SANS contrast matching, leaving only the scattering signal from Blue to be detected. Results obtained by contrast matching were confirmed by NOESY NMR-spectroscopy, showing that Blue interacts with the positively charged DTAB head groups and with up to the 4th neighbouring methylene group of the DTAB C12-alkyl chain. Its localization in the outer layer of the Blue-DTAB co-assembly explains the uniaxial growth of spheroidal DTAB micelles to wormlike micelles with increasing [Blue] : [DTAB] ratio from 0 : 1 to 1 : 3. This is in line with the concept of the packing parameter for amphiphilic substances.

Adhesion, proliferation, and detachment of various cell types on thermoresponsive microgel coatings.

Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond. On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.

Stable DOPG/Glycyrrhizin Vesicles with a Wide Range of Mixing Ratios: Structure and Stability as Seen by Scattering Experiments and Cryo-TEM.

Phosphatidylglycerols represent a large share of the lipids in the plasmamembrane of procaryotes. Therefore, this study investigates the role of charged lipids in the plasma membrane with respect to the interaction of the antiviral saponin glycyrrhizin with such membranes. Glycyrrhizin is a natural triterpenic-based surfactant found in licorice. Vesicles made of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1'-glycerol) (DOPG)/glycyrrhizin are characterized by small-angle scattering with neutrons and X-rays (SANS and SAXS). Small-angle scattering data are first evaluated by the model-independent modified Kratky-Porod method and afterwards fitted by a model describing the shape of small unilamellar vesicles (SUV) with an internal head-tail contrast. Complete miscibility of DOPG and glycyrrhizin was revealed even at a ratio of lipid:saponin of 1:1. Additional information about the chain-chain correlation distance of the lipid/saponin mixtures in the SUV structures is obtained from wide-angle X-ray scattering (WAXS).

Pepsin Digest of Gliadin Forms Spontaneously Amyloid-Like Nanostructures Influencing the Expression of Selected Pro-Inflammatory, Chemoattractant, and Apoptotic Genes in Caco-2 Cells: Implications for Gluten-Related Disorders.

SCOPE: Proteolysis-resistant gliadin peptides are intensely investigated in biomedical research relates to celiac disease and gluten-related disorders. Herein, the first integrated supramolecular investigation of pepsin-digested gliadin peptides (p-gliadin) is presented in combination with its functional behavior in the Caco-2 cell line. METHODS AND RESULTS: First, gliadins are degraded by pepsin at pH 3, and the physicochemical properties of p-gliadin are compared with gliadin. An integrated approach using interfacial, spectroscopic, and microscopic techniques reveals that the p-gliadin forms spontaneously soluble large supramolecular structures, mainly oligomers and fibrils, capable of binding amyloid-sensitive dyes. The self-assembly of p-gliadin starts at a concentration of 0.40 µg mL-1 . Second, the stimulation of Caco-2 cells with the p-gliadin supramolecular system is performed, and the mRNA expression levels of a panel of genes are tested. The experiments show that p-gliadin composed of supramolecular structures triggers significant mRNA up-regulation (p < 0.05) of pro-apoptotic biomarkers (ratio Bcl2/Bak-1), chemokines (CCL2, CCL3, CCL4, CCL5, CXCL8), and the chemokine receptor CXCR3. CONCLUSIONS: This work demonstrates that p-gliadin is interfacial active, forming spontaneously amyloid-type structures that trigger genes in the Caco-2 cell line involved in recruiting specialized immune cells.

Accounting for Cooperativity in the Thermotropic Volume Phase Transition of Smart Microgels.

A full quantitative description of the swelling of smart microgels is still problematic in many cases. The original approach of Flory and Huggins for the monomer-solvent interaction parameter χ cannot be applied to some microgels. The reason for this obviously is that the cross-linking enhances the cooperativity of the volume phase transitions, since all meshes of the network are mechanically coupled. This was ignored in previous approaches, arguing with distinct transition temperatures for different meshes to describe the continuous character of the transition of microgels. Here, we adjust the swelling curves of a series of smart microgels using the Flory-Rehner description, where the polymer-solvent interaction parameter χ is modeled by a Hill-like equation for a cooperative thermotropic transition. This leads to a very good description of all measured microgel swelling curves and yields the physically meaningful Hill parameter ν. A linear decrease of ν is found with increasing concentration of the cross-linker N,N'-methylenebisacrylamide in the microgel particles p(NIPAM), p(NNPAM), and p(NIPMAM). The linearity suggests that the Hill parameter ν corresponds to the number of water molecules per network chain that cooperatively leave the chain at the volume phase transition. Driven by entropy, ν water molecules of the solvate become cooperatively "free" and leave the polymer network.

Aescin - a natural soap for the formation of lipid nanodiscs with tunable size.

The saponin ß-aescin from the seed extract of the horse chestnut tree Aesculus hippocastanum has demonstrated a beneficial role in clinical therapy which is in part related to its strong interaction with biological membranes. In this context the present work investigates the self-assembly of nm-sized discoidal lipid nanoparticles composed of ß-aescin and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The discoidal lipid nanoparticles reassemble from small discs into larger discs, ribbons and finally stacks of sheets upon heating from gel-phase to fluid phase DMPC. The morphological transition of the lipid nano-particles is mainly triggered by the phospholipid phase state change. The final morphology depends on the phospholipid-to-saponin ratio and the actual temperature. The study is conducted by small-angle X-ray scattering (SAXS) and transmission (TEM) and freeze fracture electron microscopy (FFEM) are used to cover larger length scales. Two different models, representing a disc and ribbon-like shape are applied to the SAXS data, evaluating possible geometries and molecular mixing of the nano-particles. The stacked sheets are analysed by the Caillé theory.

Smart microgels investigated by super-resolution fluorescence microscopy: influence of the monomer structure on the particle morphology.

In a recent publication [Bergmann et al. Phys. Chem. Chem. Phys., 2018, 20, 5074-5083] we presented a method which enables to investigate the morphology of microgels by superresolution fluorescence microscopy. Here, this method is applied to three microgel species, based on N-isopropylmethacrylamide (NIPMAM), N-n-propylacrylamide (NNPAM) and N-n-propylmethacrylamide (NNPMAM)) with 5, 7.5 and 10 mol% cross-linker, respectively. Super-resolution microscopy reveals differences of the network morphology of the synthesized particles showing the importance of the monomer structure.

Suzuki-Miyaura Cross-Coupling of Bromotryptophan Derivatives at Ambient Temperature.

Mild reaction conditions are highly desirable for bio-orthogonal side chain derivatizations of amino acids, peptides or proteins due to the sensitivity of these substrates. Transition metal catalysed cross-couplings such as Suzuki-Miyaura reactions are highly versatile, but usually require unfavourable reaction conditions, in particular, when applied with aryl bromides. Ligand-free solvent-stabilised Pd-nanoparticles represent an efficient and sustainable alternative to conventional phosphine-based catalysts, because the cross-coupling can be performed at considerably lower temperature. We report on the application of such a highly reactive heterogeneous catalyst for the Suzuki-Miyaura cross-coupling of brominated tryptophan derivatives. The solvent-stabilised Pd-nanoparticles are even more efficient than the literature-known ADHP-Pd precatalyst. Interestingly, the latter also leads to the formation of quasi-homogeneous Pd-nanoparticles as the catalytic species. One advantage of our approach is the compatibility with aqueous and aerobic conditions at near-ambient temperatures and short reaction times of only 2 h. The influence of different Nα -protecting groups, boronic acids as well as the impact of different amino acid side chains in bromotryptophan-containing peptides has been studied. Notably, a surprising acceleration of the catalysis was observed when palladium-coordinating side chains were present in proximal positions.

Heating-Induced DMPC/Glycyrrhizin Bicelle-to-Vesicle Transition: A X-Ray Contrast Variation Study.

In this study, we investigated the conversion of lipid bicelles into vesicles in the case of a system composed of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the saponin glycyrrhizin in the presence of sucrose. Glycyrrhizin is a biosurfactant present in the licorice root and possesses a triterpenic hydrophobic backbone and a hydrophilic headgroup built from two sugar molecules. The aim of this study is to determine the initial bicelle size at temperatures below the lipid's main phase transition temperature Tm and, based on these results, characteristics of the temperature-induced bicelle-to-vesicle transition. Moreover, the influence of the heating rate on this transition is followed. The general picture concluded from photon correlation spectroscopy and small angle X-ray scattering was confirmed by additional imaging with cryogenic transmission electron microscopy. Small angle X-ray scattering was especially used to determine size parameters of the existing structures. To enhance the contrast for X-rays, a buffer containing 25 wt% sucrose was used. It was found that larger vesicles were formed from smaller precursor particles and that monodisperse precursors are required for formation of very monodisperse vesicles upon temperature increase. At high glycyrrhizin contents and above a critical heating rate of ∼5°C min-1, the polydispersity of these vesicles is decoupled from both parameters, glycyrrhizin content and heating rate. However, the vesicle size stays tunable by the glycyrrhizin content and increases upon increasing the glycyrrhizin concentration. Therefore, vesicles of defined size and with a rather low polydispersity of ∼12-14% can be formed.

rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations.

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme ß-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with ß-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

Hydration and Solvent Exchange Induced Swelling and Deswelling of Homogeneous Poly(N-isopropylacrylamide) Microgel Thin Films.

The investigation of the response kinetics of smart colloidal microgel films is crucial for their optimization to enable advanced applications. We study the classical thermoresponsive microgel model system N-isopropylacrylamide cross-linked with N,N'-methylenebisacrylamide. Without the typically used polyelectrolyte coating of the substrate, thin microgel films are prepared in a single spin-coating step. Atomic force microscopy measurements reveal an extremely dense packing, resulting in a homogeneous compact thin film of microgel particles. The hydration kinetics of these films in H2O and D2O atmospheres as well as the kinetics of the solvent exchange between both water species are investigated with in situ time-of-flight neutron reflectometry (TOF-NR) and in situ Fourier-transform infrared (FTIR) spectroscopy. With accounting for a nonconstant humid atmosphere, the intrinsic diffusion dynamics of water molecules into the thin microgel film are modeled and the specific time constant τ and the effective Flory-Huggins interaction parameter χeff are determined. Comparing the results in H2O and D2O atmospheres, TOF-NR and FTIR spectroscopy results show an increased affinity of the microgel films toward H2O as compared to D2O. From the FTIR spectroscopy data, we further identify different kinetics of intermolecular processes and order them according to their temporal evolution.

Aescin-Induced Conversion of Gel-Phase Lipid Membranes into Bicelle-like Lipid Nanoparticles.

Mixtures of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the saponin ß-aescin spontaneously form monodisperse, bilayered discoidal micelles (also known as "bicelles" or "nanodisks") in aqueous solution. Such bicelles form below the melting temperature of DMPC when the phospholipids are in the rigid Lß' state and are precursors of spontaneously formed vesicles. The aescin concentration must be far above the cmcaescin (≈0.3-0.4 mM). It was found that the shape and size of the bicelles are tunable by composition. High amounts of aescin decrease the size of the bicelles from diameters of ∼300 Å at 7 mol % to ∼120 Å at 30 mol % ß-aescin. The structures are scrutinized by complementary small-angle X-ray and neutron scattering experiments. The scattering curves are subsequently analyzed by a model-independent (indirect Fourier transform analysis) and a model-based approach where bicelles are described as polydisperse bilayer disks encircled by a ß-aescin rim. Moreover, the monomodal distribution and low polydispersity of the samples were confirmed by photon correlation spectroscopy. The discoidal structures were visualized by transmission electron microscopy.

Improved Smart Microgel Carriers for Catalytic Silver Nanoparticles.

Acrylamide-based, thermoresponsive core-shell microgels with a linear phase transition region are used as improved carriers for catalytically active silver nanoparticles in the present study. In this context, we investigated the swelling behavior of the carriers and the stability of the silver nanoparticles inside the polymer network with photon correlation spectroscopy, transmission electron microscopy, and by following the surface plasmon resonance of the nanoparticles. Depending on the cross-linker content of the microgel core, we observed very good stability of the nanoparticles inside the microgel network, with nearly no bleeding or aggregation of the nanoparticles over several weeks for core cross-linker contents of 5 and 10 mol %. The architecture of the hybrid particles in the swollen state was investigated with cryogenic transmission electron microscopy. The particles exhibit a core-shell structure, with the silver nanoparticles located mainly at the interface between the core and shell. This architecture was not used before and seems to grant advanced stability to the nanoparticles inside the network in combination with good switchability of the catalytic activity. This was measured by following the reduction of 4-nitrophenole, which is a well-studied model reaction. The obtained Arrhenius plots show that similar to previous works, the swelling of the core and shell can influence the catalytic activity of the silver nanoparticles. As mentioned before, the cross-linker content of the core seems to be a very important parameter for the switchability of the catalytic activity. A higher cross-linker content of the core seems to be connected to a stronger influence of the carrier swelling degree on the catalytic activity of the silver nanoparticles.

Synthesis of smart dual-responsive microgels: correlation between applied surfactants and obtained particle morphology.

Thermo- and pH-responsive copolymer microgels were obtained by surfactant-assisted precipitation polymerization of N-isopropylacrylamide (NIPAM) and acrylic acid (AAc). The surfactants used were sodium dodecylsulfate (SDS), dodecyltrimethylammonium bromide (DTAB) and the nonionic n-octyl-ß-d-glucopyranoside (C8G1). We investigate the influence of the surfactants on the acrylic acid incorporation rate, the particle size, particle morphology, and the swelling behaviour at pH 4 and pH 7, at which AAc is neutral or charged, respectively. It is shown that each surfactant has a specific influence, which is connected to its role in the polymerization mechanism and its charge. A combined FTIR and PCS study reveals that the particles undergo a temperature-induced change in microstructure, even if the particle hydrodynamic radius does not change significantly.

Temperature dependent self-organization of DMPC membranes promoted by intermediate amounts of the saponin aescin.

The plant-derived biosurfactant aescin is naturally present in many plants and is used for treatment of disorders such as varicose veins and inflammation of veins. The hemolytic activity of this saponin is attributed to its interaction with cholesterol in the red blood cell membrane. This work investigates the phase and aggregation behavior of saponin-containing model membranes consisting of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The aescin concentrations studied range from 1 mol% to 7 mol% with respect to the total lipid content. The methods of choice to elucidate the structural picture are small-angle scattering of X-rays (SAXS) and neutrons (SANS) and cryogenic transmission electron microscopy (cryo-TEM). SANS and SAXS revealed that at lower aescin contents vesicular structures are conserved and vesicles tend to aggregate already at aescin contents of around 1 mol%. Aggregation and vesicle deformation effects are found to be stronger when the phospholipids are in the L [Formula: see text] phase. With increasing aescin content, mixed structures, i.e. aggregated and deformed vesicles and solubilized bilayer fragments, are present. This was proven for a sample with 4 mol% aescin by cryo-TEM. An increasing aescin amount leads to membrane decomposition and free standing bilayers which tend to build stacks at high temperature. These stacks are characterized by SAXS using the modified Caillé theory. Analyses and model dependent fitting reveal formation of well-defined structures beginning at 7 mol% aescin.

Volume phase transition kinetics of smart N-n-propylacrylamide microgels studied by time-resolved pressure jump small angle neutron scattering.

The use of smart colloidal microgels for advanced applications critically depends on their response kinetics. We use pressure jump small angle neutron scattering with supreme time resolution to study the rapid volume phase transition kinetics of such microgels. Utilizing the pressure induced microphase separation inside the microgels we were able to resolve their collapse and swelling kinetics. While the collapse occurs on a time scale of 10 ms, the particle swelling turned out to be much faster. Photon correlation spectroscopy and static small angle neutron scattering unambiguously show, that the much slower collapse can be associated with the complex particle architecture exhibiting a loosely-crosslinked outer region and a denser inner core region. These insights into the kinetics of stimuli-responsive materials are of high relevance for their applications as nano-actuators, sensors or drug carriers. Moreover, the used refined pressure jump small angle neutron scattering technique is of broad interest for soft matter studies.