Solvent Dependent Folding of an Amphiphilic Polyaramid.

A series of synthetic alternating and amphiphilic aromatic amide polymers were synthesized by a step growth polymerization. Alternating meta- and para-linkages were introduced to force the polymer chain into a helical shape in the highly polar solvent water. The polymers were analyzed by 1H NMR spectroscopy and SEC in polar aprotic solvents such as DMSO and DMF. However, the polymers also showed good solubility in water. 1H NMR spectroscopy, small-angle X-ray scattering, and dynamic light scattering provided clear evidence of polymer folding in water but not DMF. We employed parallel tempering metadynamics in the well-tempered ensemble (PTMetaD-WTE) to simulate the free energy surfaces of an analogous model polymer in DMF and water. The simulations gave a molecular model of an unfolded structure in DMF and a helically folded tubular structure in water.

Human Antimicrobial Peptide Triggered Colloidal Transformations in Bacteria Membrane Lipopolysaccharides.

Growing concerns of bacterial resistance against conventional antibiotics shifts the research focus toward antimicrobial peptide (AMP)-based materials. Most AMPs kill gram-negative bacteria by destroying their inner membrane, but have to first pass the outer membrane covered with lipopolysaccharides (LPS). Their interplay with the LPS is crucial for bactericidal activity, but is yet to be elucidated in detail. In this study, self-assemblies of Escherichia coli LPS with the human cathelicidin AMP LL-37, free and encapsulated into glyceryl monooleate (GMO) lipid nanoparticles, are analyzed using synchrotron small angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy. Circular dichroism spectroscopy is used to study modifications in LL-37's secondary structure. LPS is found to form elongated micelles and the addition of LL-37 induces their transformation to multilamellar structures. LPS' addition to GMO cubosomes triggers the swelling of the internal cubic structure, while in multilamellar GMO/LL-37 nanocarriers it causes transitions into unstructured particles. The insights on the interactions among LPS and LL-37, in its free form or encapsulated in GMO dispersions, may guide the design of LPS-responsive antimicrobial nanocarriers. The findings may further assist the formulation of antimicrobial nanomaterials with enhanced penetration of LPS layers for improved destruction of bacterial membranes.

Donor-Acceptor Stenhouse Adduct-Polydimethylsiloxane-Conjugates for Enhanced Photoswitching in Bulk Polymers.

Donor-acceptor Stenhouse adducts (DASAs) are a rapidly emerging class of visible light-activated photochromes and DASA-functionalized polymers hold great promise as biocompatible photoresponsive materials. However, the photoswitching performance of DASAs in solid polymer matrices is often low, particularly in materials below their glass transition temperature. To overcome this limitation, DASAs are conjugated to polydimethylsiloxanes which have a glass transition temperature far below room temperature and which can create a mobile molecular environment around the DASAs for achieving more solution-like photoswitching kinetics in bulk polymers. The dispersion of DASAs conjugated to such flexible oligomers into solid polymer matrices allows for more effective and tunable DASA photoswitching in stiff polymers, such as poly(methyl methacrylate), without requiring modifications of the matrix. The photoswitching of conjugates with varying polymer molecular weight, linker type, and architecture is characterized via time-dependent UV-vis spectroscopy in organic solvents and blended into polymethacrylate films. In addition, DASA-functionalized polydimethylsiloxane networks, accessible via the same synthetic route, provide an alternative solution for achieving fast and efficient DASA photoswitching in the bulk owing to their intrinsic softness and flexibility. These findings may contribute to the development of DASA-functionalized materials with better tunable, more effective, and more reversible modulation of their optical properties.

(Anti)viral Material Design Guided by Scattering Methods.

Viruses are nature's own nanoparticles that are highly symmetric and monodisperse in size and shape with well-defined surface chemistry. They have evolved for optimal cell interactions, genetic information delivery and replication by the host cell over millions of years. These features render them into very efficient pathogens that place a severe burden onto the health of our society. At the same time, they are highly interesting objects for colloidal studies and building blocks for advanced bio-inspired materials for health applications. Their characterisation requires sophisticated experimental techniques such as scattering of X-rays, neutrons, and light to probe structures and interactions from the nanometre to the micrometre length-scale in solution. This contribution summarizes the recent progress in the field of virus self-assembly and virus-based biopolymer composites for advanced material design. It discusses the advances and highlights some of the challenges in the characterization of structure and dynamics in these materials with a focus on scattering techniques. It further demonstrates selected applications in the field of food and water purification.

Virus pH-Dependent Interactions with Cationically Modified Cellulose and Their Application in Water Filtration.

Norovirus and Rotavirus are among the pathogens causing a large number of disease outbreaks due to contaminated water. These viruses are nanoscale particles that are difficult to remove by common filtration approaches which are based on physical size exclusion, and require adsorption-based filtration methods. This study reports the pH-responsive interactions of viruses with cationic-modified nanocellulose and demonstrates a filter material that adsorbs nanoscale viruses and can be regenerated by changing the solution's pH. The bacteria viruses Qbeta and MS2, with diameters below 30 nm but different surface properties, are used to evaluate the pH-dependency of the interactions and the filtration process. Small angle X-ray scattering, cryogenic transmission electron microscopy, and ζ-potential measurements are used to study the interactions and analyze changes in their nanostructure and surface properties of the virus upon adsorption. The virus removal capacity of the cationic cellulose-based aerogel filter is 99.9% for MS2 and 93.6% for Qbeta, at pH = 7.0; and desorption of mostly intact viruses occurs at pH = 3.0. The results contribute to the fundamental understanding of pH-triggered virus-nanocellulose self-assembly and can guide the design of sustainable and environmentally friendly adsorption-based virus filter materials as well as phage and virus-based materials.

Design and Characterization of Bio-inspired Antimicrobial Nanomaterials.

Colloidal structures are crucial components in biological systems and provide a vivid and seemingly infinite source of inspiration for the design of functional bio-inspired materials. They form multi-dimensional confinements and shape living matter, and transport and protect bioactive molecules in harsh biological environments such as the stomach. Recently, colloidal nanostructures based on natural antimicrobial peptides have emerged as promising alternatives to conventional antibiotics. This contribution summarizes the recent progress in the understanding and design of these bio-inspired antimicrobial nanomaterials, and discusses their advances in the form of dispersions and as surface coatings. Their potential for applications in future food and healthcare materials is also highlighted. Further, it discusses challenges in the characterization of structure and dynamics in these materials.

pH-Responsive Nano-Self-Assemblies of the Anticancer Drug 2-Hydroxyoleic Acid.

pH-responsive lipid nanocarriers have the potential to selectively target the acidic extracellular pH environment of cancer tissues and may further improve the efficacy of chemotherapeutics by minimizing their toxic side-effects. Here, we present the design and characterization of pH-sensitive nano-self-assemblies of the poorly water-soluble anticancer drug 2-hydroxyoleic acid (2OHOA) with glycerol monooleate (GMO). pH-triggered nanostructural transformations from 2OHOA/GMO nanoparticles with an internal inverse hexagonal structure (hexosomes) at pH around 2.0-3.0, via nanocarriers with an internal inverse bicontinuous cubic structure (cubosomes) at pH 2.0-4.5, to vesicles at pH 4.5-7.4 were observed with synchrotron small-angle X-ray scattering, and cryogenic transmission electron microscopy. ζ-potential measurements highlight that the pH-driven deprotonation of the carboxylic group of 2OHOA, and the resulting charge-repulsions at the lipid-water interface account for these nanostructural alterations. The study provides detailed insight into the pH-dependent self-assembly of 2OHOA with GMO in excess buffer at physiologically relevant pH values, and discusses the effects of pH alterations on modulating their nanostructure. The results may guide the further development of pH-responsive anticancer nanocarriers for the targeted delivery of chemotherapeutics to the local microenvironment of tumor cells.

Structural characterization of self-assemblies of new omega-3 lipids: docosahexaenoic acid and docosapentaenoic acid monoglycerides.

The attractiveness of new omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides (MAGs) lies in the amphiphilic nature and the beneficial health effects as PUFA precursors in various disorders including cancer, pulmonary hypertension, and inflammatory diseases. For exploring the potential therapeutic applications of these new amphiphilic lipids, particularly as main lipid constituents in the development of nanocarriers for delivery of drugs and PUFAs, it is of paramount importance to gain insight into their self-assembly behavior on exposure to excess water. This work describes the structural characteristics of self-assemblies based on two newly synthesized MAGs, namely docosahexaenoic acid (MAG-DHA) and docosapentaenoic acid (MAG-DPA) monoglycerides, on exposure to excess water. We found that both lipids tend to form a dominant inverse hexagonal (H2) phase in excess water at 25 °C and a temperature-triggered structural transition to an inverse micellar solution (L2 phase) is detected similar to that recently reported (A. Yaghmur et al., Langmuir, 2017, 33, 14045-14057) for eicosapentaenoic acid monoglyceride (MAG-EPA). An experimental SAXS structural evaluation study on the temperature-dependent behavior of these new monoglycerides is provided, and the effects of unsaturation degree and fatty acyl chain length on the self-assembled structural features in excess water and on the H2-L2 phase transition temperature are discussed. In addition, hexosomes stabilized by using the triblock copolymer F127 and the food-grade emulsifier citrem were investigated to gain insights into the effects of stabilizer and temperature on the internal nanostructure. These nanoparticles are attractive for use in the development of nanocarriers for delivering drugs and/or nutritional compounds as the beneficial health effects of ω-3 PUFA monoglycerides can be combined with those of loaded therapeutic agents or nutraceuticals.

Enzyme-Triggered Dissociation of a FRET-Based Protein Biosensor Monitored by Synchrotron SAXS.

Protein biosensors are widely used for the monitoring of metabolite concentration and enzymatic activities inside living cells and in in vitro applications. Neutrophil elastase (NE) is a serine protease of relevance in inflammatory diseases whose activity can lead to pathological conditions if unregulated. This study focuses on the structural characterization of a biosensor for NE activity based on Förster resonance energy transfer (FRET). The cleavage by NE results in dissociation of the FRET fluorescent protein pair and alteration of the fluorescent emission spectrum. We have used small angle x-ray scattering at a high intensity synchrotron source, combined with model-free analysis of the scattering data, to demonstrate the structure of the biosensor and the effect of its exposure to NE on size and shape. These investigations, together with biochemical studies, established the nanostructure-activity relationship that may contribute to the detailed understanding of the FRET-based biosensor and guide the rational design of new biosensor constructs.

Structural Investigation of Bulk and Dispersed Inverse Lyotropic Hexagonal Liquid Crystalline Phases of Eicosapentaenoic Acid Monoglyceride.

Recent studies demonstrated the potential therapeutic use of newly synthesized omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides owing to their beneficial health effects in various disorders including cancer and inflammation diseases. To date, the research was mainly focused on exploring the biological effects of these functional lipids. However, to the best of our knowledge, there is no report on the hydration-mediated self assembly of these lipids that leads to the formation of nanostructures, which are attractive for use as vehicles for the delivery of drugs and functional foods. In the present study, we investigated the temperature-composition phase behaviour of eicosapentaenoic acid monoglyceride (MAG-EPA), which is one of the most investigated ω-3 PUFA monoglycerides, during a heating-cooling cycle in the temperature range of 5-60 °C. Experimental synchrotron small-angle X-ray scattering (SAXS) evidence on the formation of a dominant inverse hexagonal (H2) lyotropic liquid crystalline phase and its temperature-induced transition to an inverse micellar solution (L2 phase) is presented for the fully hydrated bulk MAG-EPA system and its corresponding dispersion. We produced colloidal MAG-EPA hexosomes with an internal inverse hexagonal (H2) lyotropic crystalline phase in the presence of F127, a well-known polymeric stabilizer, or citrem, which is a negatively charged food-grade emulsifier. In this work, we report also on the formation of MAG-EPA hexosomes by vortexing MAG-EPA in excess aqueous medium containing F127 at room temperature. This low-energy emulsification method is different than most reported studies in the literature that have demonstrated the need for using a high-energy input during the emulsification step or adding an organic solvent for the formation of such colloidal nonlamellar liquid crystalline dispersions. The designed nanoparticles hold promise for future drug and functional food delivery applications due to their unique structural properties and the potential health-promoting effects of MAG-EPA.

Softwood Lignin Self-Assembly for Nanomaterial Design.

Lignin is a natural, renewable biopolymer synthesized by plants. It is a macromolecule consisting of aromatic structures with high density of functional groups making it an ideal precursor for the design of sustainable biomaterials for applications such as drug delivery. The rational design of these materials requires an in-depth understanding of the underlying lignin self-assembly in solution. Colloidal transformations from nanosized lignin assemblies to submicron-sized spherical particles upon solvent exchange were studied using small-angle X-ray scattering, dynamic light scattering, and electron microscopy. The surface fractal structure and stability of these particles was found to be strongly solvent and pH dependent, with aggregation to a gel-like material at low pH. The results may have important implications for the design of nanostructured lignin-based functional materials for consideration in various fields such as food science and biomedicine.

Self-coacervation of modular squid beak proteins - a comparative study.

The beak of the Humboldt squid is a biocomposite material made solely of organic components - chitin and proteins - which exhibits 200-fold stiffness and hardness gradients from the soft base to the exceptionally hard tip (rostrum). The outstanding mechanical properties of the squid beak are achieved via controlled hydration and impregnation of the chitin-based scaffold by protein coacervates. Molecular-based understanding of these proteins is essential to mimic the natural beak material. Here, we present detailed studies of two histidine-rich beak proteins (HBP-1 and -2) that play central roles during beak bio-fabrication. We show that both proteins have the ability to self-coacervate, which is governed intrinsically by the sequence modularity of their C-terminus and extrinsically by pH and ionic strength. We demonstrate that HBPs possess dynamic structures in solution and achieve maximum folding in the coacervate state, and propose that their self-coacervation is driven by hydrophobic interactions following charge neutralization through salt-screening. Finally, we show that subtle differences in the modular repeats of HBPs result in significant changes in the rheological response of the coacervates. This knowledge may be exploited to design self-coacervating polypeptides for a wide range of engineering and biomedical applications, for example bio-inspired composite materials, smart hydrogels and adhesives, and biomedical implants.

Microemulsions as Potential Carriers of Nisin: Effect of Composition on Structure and Efficacy.

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

Understanding the Mechanism of Enzyme-Induced Formation of Lyotropic Liquid Crystalline Nanoparticles.

Liquid crystalline nanoparticles have shown great potential for application in fields of drug delivery and agriculture. However, optimized approaches to generating these dispersions have long been sought after. This study focused on understanding the mechanism of formation of cubosomes during the recently reported enzymatic approach and extending the approach to alternative lipid types other than phytantriol. The chain length of digestible lipids was found to influence the effectiveness of triglycerides in disrupting the equilibrium cubic phase structure to form the emulsion precursor. In general, a greater hydrophobicity of the triglyceride required a lower concentration to inhibit liquid crystal structure formation. Selachyl alcohol was also examined due to its nondigestible trait and ability to form the inverted hexagonal phase. Digestion of its precursor emulsion formed hexosomes analogous to the phytantriol-based systems. Finally, the assumption that fatty acids liberated during digestion needed to partition out of the nondigestible lipids for the re-formation of the phase structure was found to be untrue. Their ionization state, however, did have an effect on the resulting nanostructure, and this unique property could potentially provide a useful attribute for oral drug delivery systems.

Phospholipid-based self-assembled mesophase systems for light-activated drug delivery.

The manipulation of the structure of phospholipid-based mesophases to induce a slow to fast drug release profile has potential for use in therapeutic situations where continuous absorption of drug is not desirable and reduce the frequency of injection for short acting or rapidly cleared drugs in treatments for diseases such as macular degeneration. This study had two aims; firstly to confirm the phase behaviour of 20 mol% cholesterol in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), which was previously reported to transition from lamellar (slow release) to bicontinuous cubic (fast release) phase with increasing temperature. Contrary to the literature, no bicontinuous cubic phase was observed but a transition to the inverse hexagonal phase occurred at all POPE : cholesterol ratios investigated. The second aim was to render these mesophases responsive to near-infrared laser (NIR) irradiation by incorporation of gold nanorods (GNR) incorporated into the POPE system to induce photothermal heating. The inclusion of 3 nM GNR in POPE systems induced reversible disruption of lipid packing equivalent to increasing the temperature to 55 °C when irradiated for 30 s. This study confirmed that although the previously published phase behavior was not correct, GNR and NIR can be used to manipulate the self-assembled mesophases in phospholipid-based systems and highlights the potential for a phospholipid-based light-activated drug delivery system.

Self-assembly structure formation during the digestion of human breast milk.

An infant's complete diet, human breast milk, is the basis for its survival and development. It contains water-soluble and poorly water-soluble bioactive components, metabolic messages, and energy, all of which are made bioavailable during the digestion process in the infant's gastrointestinal tract. Reported is the first discovery of highly geometrically organized structures formed during the digestion of human breast milk under simulated in vivo conditions using small-angle X-ray scattering and cryogenic transmission electron microscopy. Time of digestion, pH, and bile salt concentration were found to have symbiotic effects gradually tuning the oil-based environment inside the breast milk globules to more water-like structures with high internal surface area. The structure formation is necessarily linked to its function as carriers for poorly water-soluble molecules in the digestive tract of the infant.

pH-driven colloidal transformations based on the vasoactive drug nicergoline.

The structure of colloidal self-assembled drug delivery systems can be influenced by intermolecular interactions between drug and amphiphilic molecules, and is important to understand in the context of designing improved delivery systems. Controlling these structures can enable controlled or targeted release systems for poorly water-soluble drugs. Here we present the interaction of the hydrophobic vasoactive drug nicergoline with the internal structure of nanostructured emulsion particles based on the monoglyceride-water system. Addition of this drug leads to modification of the internal bicontinuous cubic structure to generate highly pH-responsive systems. The colloidal structures were characterized with small-angle X-ray scattering and visualized using cryogenic transmission electron microscopy. Reversible transformations to inverse micelles at high pH, vesicles at low pH, and the modification of the spacing of the bicontinuous cubic structure at intermediate pH were observed, and enabled the in situ determination of an apparent pKa for the drug in this system--a difficult task using solution-based approaches. The characterization of this phase behavior is also highly interesting for the design of pH-responsive controlled release systems for poorly water-soluble drug molecules.

pH-responsive micelles based on caprylic acid.

Free fatty acids play a vital role as fuel for cells and in lipid metabolism. During lipid digestion in the gastrointestinal tract, triglycerides are hydrolyzed, resulting in free fatty acid and monoglyceride amphiphilic products. These components, together with bile salts, are responsible for the transport of lipids and poorly water-soluble nutrients and xenobiotics from the intestine into the circulatory system of the body. In this study, we show that the self-assembly of digestion products from medium-chain triglycerides (tricaprylin) in combination with bile salt and phospholipid is highly pH-responsive. Individual building blocks of caprylic acid within the mixed colloidal structures are mapped using a combination of small-angle X-ray and neutron scattering combined with both solvent contrast variation and selective deuteration. Modeling of the scattering data shows transitions in the size and shape of the micelles in combination with a transfer of the caprylic acid from the core of the micelles to the shell or into the bulk water upon increasing pH. The results help to understand the process of lipid digestion with a focus on colloidal structure formation and transformation for the delivery of triglyceride lipids and other hydrophobic functional molecules.

Generation of geometrically ordered lipid-based liquid-crystalline nanoparticles using biologically relevant enzymatic processing.

High-symmetry lipid nanoparticles with internal bicontinuous cubic phase structure (cubosomes) are prepared from a simple emulsion containing a mixture of a nondigestible lipid (phytantriol) and a digestible short-chained triglyceride using enzymatic lipolysis of the incorporated short-chained triglyceride. The lipolytic products partition away from the nondigestible lipid, resulting in crystallization of the cubic-phase internal structure. Time-resolved small-angle X-ray scattering revealed the kinetics of the disorder-to-order transition, with cryo-transmission electron microscopy showing an absence of liposomes. The new approach offers a new "sideways" method for the generation of lipid-based nanostructured materials that avoids the problems of top-down and bottom-up approaches.

Biocompatible Rhamnolipid Self-Assemblies with pH-Responsive Antimicrobial Activity.

There is an urgent need for alternative antimicrobial materials due to the growing challenge of bacteria becoming resistant to conventional antibiotics. This study demonstrates the creation of a biocompatible pH-switchable antimicrobial material by combining bacteria-derived rhamnolipids (RL) and food-grade glycerol monooleate (GMO). The integration of RL into dispersed GMO particles, with an inverse-type liquid crystalline cubic structure in the core, leads to colloidally stable supramolecular materials. The composition and pH-triggered structural transformations are studied with small-angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering. The composition-structure-activity relationship is analyzed and optimized to target bacteria at acidic pH values of acute wounds. The new RL/GMO dispersions reduce Staphylococcus aureus (S. aureus) populations by 7-log after 24 h of treatment with 64 µg mL-1 of RL and prevent biofilm formation at pH = 5.0, but have no activity at pH = 7.0. Additionally, the system is active against methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration of 128 µg mL-1 at pH 5.0. No activity is found against several Gram-negative bacteria at pH 5.0 and 7.0. The results provide a fundamental understanding of lipid self-assembly and the design of lipid-based biomaterials, which can further guide the development of alternative bio-based solutions to combat bacteria.