Dynamics of Single-Chain Nanoparticles under Crowding: A Neutron Spin Echo Study.

We present a neutron spin echo (NSE) investigation to examine the impact of macromolecular crowding on the dynamics of single-chain nanoparticles (SCNPs), serving as synthetic models for biomacromolecules with flexibility and internal degrees of freedom, such as intrinsically disordered proteins (IDPs). In particular, we studied the dynamics of a medium-size poly(methyl methacrylate) (PMMA)-based SCNP (33 kDa) in solutions with low- (10 kDa) and high- (100 kDa) molecular weight analogous deuterated PMMA linear crowders. The dynamic structure factors of the SCNPs in dilute solution show certain degrees of freedom, yet the analysis in terms of the Zimm model reveals high internal friction that effectively stiffens the chain-a phenomenon also observed for IDPs. Under crowding conditions, the internal dynamics remains essentially unchanged, but the center-of-mass diffusion slows down. The effective viscosity felt by the SCNPs at the timescales probed by NSE is lower than the macroscopic viscosity of the crowder solution, and it does not depend significantly on the molecular weight.

Structure of Single-Chain Nanoparticles under Crowding Conditions: A Random Phase Approximation Approach.

The conformation of poly(methyl methacrylate) (PMMA)-based single-chain nanoparticles (SCNPs) and their corresponding linear precursors in the presence of deuterated linear PMMA in deuterated dimethylformamide (DMF) solutions has been studied by small-angle neutron scattering (SANS). The SANS profiles were analyzed in terms of a three-component random phase approximation (RPA) model. The RPA approach described well the scattering profiles in dilute and crowded solutions. Considering all the contributions of the RPA leads to an accurate estimation of the single chain form factor parameters and the Flory-Huggins interaction parameter between PMMA and DMF. The value of the latter in the dilute regime indicates that the precursors and the SCNPs are in good solvent conditions, while in crowding conditions, the polymer becomes less soluble.

Neat Protein Single-Chain Nanoparticles from Partially Denatured BSA.

The main challenge for the preparation of protein single-chain nanoparticles (SCNPs) is the natural complexity of these macromolecules. Herein, we report the suitable conditions to produce "neat" bovine serum albumin (BSA) single-chain nanoparticles (SCNPs) from partially denatured BSA, which involves denaturation in urea and intramolecular cross-linking below the overlap concentration. We use two disuccinimide ester linkers containing three and six methylene spacer groups: disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS), respectively. Remarkably, the degree of internal cross-linking can be followed simply and efficiently via 1H NMR spectroscopy. The associated structural changes-as probed by small-angle neutron scattering (SANS)-reveal that the denatured protein has a random-like coil conformation, which progressively shrinks with the addition of DSG or DSS, thus allowing for size control of the BSA-SCNPs with radii of gyration down to 5.4 nm. The longer cross-linker exhibits slightly more efficiency in chain compaction with a somewhat stronger size reduction but similar reactivity at a given cross-linker concentration. This reliable method is applicable to a wide range of compact proteins since most proteins have appropriate reactive amino acids and denature in urea. Critically, this work paves the way to the synthesis of "neat", biodegradable protein SCNPs for a range of applications including nanomedicine.

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1.

The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.

Dynamic Processes and Mechanisms Involved in Relaxations of Single-Chain Nano-Particle Melts.

We present a combined study by quasielastic neutron scattering (QENS), dielectric and mechanical spectroscopy, calorimetry and wide-angle X-ray diffraction on single-chain nano-particles (SCNPs), using the corresponding linear precursor chains as reference, to elucidate the impact of internal bonds involving bulky cross-links on the properties of polymer melts. Internal cross-links do not appreciably alter local properties and fast dynamics. This is the case of the average inter-molecular distances, the ß-relaxation and the extent of the atomic displacements at timescales faster than some picoseconds. Contrarily, the α-relaxation is slowed down with respect to the linear precursor, as detected by DSC, dielectric spectroscopy and QENS. QENS has also resolved broader response functions and stronger deviations from Gaussian behavior in the SCNPs melt, hinting at additional heterogeneities. The rheological properties are also clearly affected by internal cross-links. We discuss these results together with those previously reported on the deuterated counterpart samples and on SCNPs obtained through a different synthesis route to discern the effect of the nature of the cross-links on the modification of the diverse properties of the melts.

Direct Observation of Dynamic Tube Dilation in Entangled Polymer Blends: A Combination of Neutron Scattering and Dielectric Techniques.

We report a microscopic observation of the time-dependent dynamic tube dilation process on isofrictional bidisperse melts. By applying neutron spin echo (NSE) and dielectric techniques on blends of long polyisoprene (PI) chains with short PI additives with different topology, we access the dynamics of the tube dilation process on a molecular scale. The time-dependent tube dilation is directly revealed by NSE as an additional time dependence of the dynamic structure factor in the local reptation regime. We identify the characteristic time of tube dilation as the terminal time of the additive.

Facile Access to Completely Deuterated Single-Chain Nanoparticles Enabled by Intramolecular Azide Photodecomposition.

Access to completely deuterated single-chain nanoparticles (dSCNPs) has remained an unresolved issue. Herein, the first facile and efficient procedure to produce dSCNPs is reported, which comprises: i) the use of commercially available perdeuterated cyclic ether monomers as starting reagents, ii) a ring-opening copolymerization process performed in bulk to produce a neat dSCNP precursor, iii) a standard azidation reaction to decorate this precursor with azide moieties, and iv) a facile intramolecular azide photodecomposition step carried out under UV irradiation at high dilution providing with highly valuable, completely deuterated soft nano-objects from the precursor. dSCNPs are used to investigate by means of neutron-scattering measurements the form factor (radius of gyration, scaling exponent) of polyethylene oxide (PEO) chains in nanocomposites with different amounts of dSCNPs. Moreover, to illustrate the possibilities offered by the synthetic route disclosed in this communication for potential applications, the significant reduction in viscosity observed in a pure melt of polyether-based single-chain nanoparticles when compared to a melt of the corresponding linear polymer chains is shown.

Controlling Complex Nanoemulsion Morphology Using Asymmetric Cosurfactants for the Preparation of Polymer Nanocapsules.

Complex nanoemulsions, comprising multiphase nanoscale droplets, hold considerable potential advantages as vehicles for encapsulation and delivery as well as templates for nanoparticle synthesis. Although methods exist to controllably produce complex emulsions on the microscale, very few methods exist to produce them on the nanoscale. Here, we examine a recently developed method involving a combination of high-energy emulsification with conventional cosurfactants to produce oil-water-oil (O/W/O) complex nanoemulsions. Specifically, we study in detail how the composition of conventional ethoxylated cosurfactants Span80 and Tween20 influences the morphology and structure of the resulting complex nanoemulsions in the water-cyclohexane system. Using a combination of small-angle neutron scattering and cryo-electron microscopy, we find that the cosurfactant composition controls the generation of complex droplet morphologies including core-shell and multicore-shell O/W/O nanodroplets, resulting in an effective state diagram for the selection of nanoemulsion morphology. Additionally, the cosurfactant composition can be used to control the thickness of the water shell contained within the complex nanodroplets. We hypothesize that this degree of control, despite the highly nonequilibrium nature of the nanoemulsions, is ultimately determined by a competition between the opposing spontaneous curvature of the two cosurfactants, which strongly influences the interfacial curvature of the nanodroplets as a result of their ultralow interfacial tension. This is supported by a correlation between cosurfactant compositions that produces complex nanoemulsions and those that produce homogeneous mixed micelles in equilibrium surfactant-cyclohexane solutions. Ultimately, we show that the formation of complex O/W/O nanoemulsions is weakly perturbed upon the addition of hydrophilic polymer precursors, facilitating their use as templates for the formation of polymer nanocapsules.

Investigation of the dynamics of aqueous proline solutions using neutron scattering and molecular dynamics simulations.

We applied quasielastic neutron scattering (QENS) techniques to samples with two different contrasts (deuterated solute/hydrogenated solvent and the opposite label) to selectively study the component dynamics of proline/water solutions. Results on diluted and concentrated solutions (31 and 6 water molecules/proline molecule, respectively) were analyzed in terms of the susceptibility and considering a recently proposed model for water dynamics [Arbe et al., Phys. Rev. Lett., 2016, 117, 185501] which includes vibrations and the convolution of localized motions and diffusion. We found that proline molecules not only reduce the average diffusion coefficient of water but also extend the time/frequency range of the crossover region ('cage') between the vibrations and purely diffusive behavior. For the high proline concentration we also found experimental evidence of water heterogeneous dynamics and a distribution of diffusion coefficients. Complementary molecular dynamics simulations show that water molecules start to perform rotational diffusion when they escape the cage regime but before the purely diffusive behavior is established. The rotational diffusion regime is also retarded by the presence of proline molecules. On the other hand, a strong coupling between proline and water diffusive dynamics which persists with decreasing temperature is directly observed using QENS. Not only are the temperature dependences of the diffusion coefficients of both components the same, but their absolute values also approach each other with increasing proline concentration. We compared our results with those reported using other techniques, in particular using dielectric spectroscopy (DS). A simple approach based on molecular hydrodynamics and a molecular treatment of DS allows rationalizing the a priori puzzling inconsistency between QENS and dielectric results regarding the dynamic coupling of the two components. The interpretation proposed is based on general grounds and therefore should be applicable to other biomolecular solutions.

Synthesis of Oil-Laden Poly(ethylene glycol) Diacrylate Hydrogel Nanocapsules from Double Nanoemulsions.

Multiple emulsions have received great interest due to their ability to be used as templates for the production of multicompartment particles for a variety of applications. However, scaling these complex droplets to nanoscale dimensions has been a challenge due to limitations on their fabrication methods. Here, we report the development of oil-in-water-in-oil (O1/W/O2) double nanoemulsions via a two-step high-energy method and their use as templates for complex nanogels comprised of inner oil droplets encapsulated within a hydrogel matrix. Using a combination of characterization methods, we determine how the properties of the nanogels are controlled by the size, stability, internal morphology, and chemical composition of the nanoemulsion templates from which they are formed. This allows for identification of compositional and emulsification parameters that can be used to optimize the size and oil encapsulation efficiency of the nanogels. Our templating method produces oil-laden nanogels with high oil encapsulation efficiencies and average diameters of 200-300 nm. In addition, we demonstrate the versatility of the system by varying the types of inner oil, the hydrogel chemistry, the amount of inner oil, and the hydrogel network cross-link density. These nontoxic oil-laden nanogels have potential applications in food, pharmaceutical, and cosmetic formulations.

Gels Obtained by Colloidal Self-Assembly of Amphiphilic Molecules.

Gelation in water-based systems can be achieved in many different ways. This review focusses on ways that are based on self-assembly, i.e., a bottom-up approach. Self-assembly naturally requires amphiphilic molecules and accordingly the systems described here are based on surfactants and to some extent also on amphiphilic copolymers. In this review we are interested in cases of low and moderate concentrations of amphiphilic material employed to form hydrogels. Self-assembly allows for various approaches to achieve gelation. One of them is via increasing the effective volume fraction by encapsulating solvent, as in vesicles. Vesicles can be constructed in various morphologies and the different cases are discussed here. However, also the formation of very elongated worm-like micelles can lead to gelation, provided the structural relaxation times of these systems is long enough. Alternatively, one may employ amphiphilic copolymers of hydrophobically modified water soluble polymers that allow for network formation in solution by self-assembly due to having several hydrophobic modifications per polymer. Finally, one may combine such polymers with surfactant self-assemblies and thereby produce interconnected hybrid network systems with corresponding gel-like properties. As seen here there is a number of conceptually different approaches to achieve gelation by self-assembly and they may even become combined for further variation of the properties. These different approaches are described in this review to yield a comprehensive overview regarding the options for achieving gel formation by self-assembly.

Oil-in-Water-in-Oil Multinanoemulsions for Templating Complex Nanoparticles.

Complex nanoemulsions involving nanodroplets with a defined inner structure have great potential for encapsulation and templating applications. We report a method to form novel complex oil-in-water-in-oil nanoemulsions using a combination of high-energy processing with mixed nonionic surfactants that simultaneously achieve ultralow interfacial tension and frustrated curvature of the water-oil interface. The method produces multinanoemulsions possessing morphologies resembling water-swollen reverse vesicles with core-shell and multicore-shell morphologies of water in cyclohexane. A combination of macroscopic and microscopic characterization conclusively verifies and quantifies the complex morphologies, which vary systematically and reproducibly with water content for water volume fractions between 0.01 and 0.10. The complex morphologies are stable tens of hours, providing a route for their use as liquid templates for internally structured nanoparticles. As a demonstration, we test the complex nanoemulsions' ability to template complex polymer nanogels.

The microscopic network structure of mussel (Mytilus) adhesive plaques.

Marine mussels of the genus Mytilus live in the hostile intertidal zone, attached to rocks, bio-fouled surfaces and each other via collagen-rich threads ending in adhesive pads, the plaques. Plaques adhere in salty, alkaline seawater, withstanding waves and tidal currents. Each plaque requires a force of several newtons to detach. Although the molecular composition of the plaques has been well studied, a complete understanding of supra-molecular plaque architecture and its role in maintaining adhesive strength remains elusive. Here, electron microscopy and neutron scattering studies of plaques harvested from Mytilus californianus and Mytilus galloprovincialis reveal a complex network structure reminiscent of structural foams. Two characteristic length scales are observed characterizing a dense meshwork (approx. 100 nm) with large interpenetrating pores (approx. 1 µm). The network withstands chemical denaturation, indicating significant cross-linking. Plaques formed at lower temperatures have finer network struts, from which we hypothesize a kinetically controlled formation mechanism. When mussels are induced to create plaques, the resulting structure lacks a well-defined network architecture, showcasing the importance of processing over self-assembly. Together, these new data provide essential insight into plaque structure and formation and set the foundation to understand the role of plaque structure in stress distribution and toughening in natural and biomimetic materials.

Oil-in-water microemulsion droplets of TDMAO/decane interconnected by the telechelic C18-EO150-C18: clustering and network formation.

The effect of a doubly hydrophobically end-capped water soluble polymer (C18-PEO150-C18) on the properties of an oil-in-water (O/W) droplet microemulsion (R ∼ 2.85 nm) has been studied as a function of the amount of added telechelic polymer. Macroscopically one observes a substantial increase of viscosity once a concentration of ∼5 hydrophobic stickers per droplet is surpassed and effective cross-linking of the droplets takes place. SANS measurements show that the size of the individual droplets is not affected by the polymer addition but it induces attractive interactions at low concentration and repulsive ones at high polymer content. Measurements of the diffusion coefficient by DLS and FCS show increasing sizes at low polymer addition that can be attributed to the formation of clusters of microemulsion droplets interconnected by the polymer. At higher polymer content the network formation leads to an additional slow relaxation mode in DLS that can be related to the rheological behaviour, while the self-diffusion observed in FCS attains a lower plateau value, i.e., the microemulsion droplets remain effectively fixed within the network. The combination of SANS, DLS, and FCS allows us to derive a self-consistent picture of the evolution of structure and dynamics of the mixed system microemulsion/telechelic polymer as a function of the polymer content, which is not only relevant for controlling the macroscopic rheological properties but also with respect to the internal dynamics as it is, for instance, relevant for the release and transport of active agents.

Self-recovering stimuli-responsive macrocycle-equipped supramolecular ionogels with unusual mechanical properties.

A chiral, crown-ether-functionalized bisurea gelator forms supramolecular gels in ionic liquids. The resulting ionogels show a remarkably high thermal stability with gel-sol transition temperatures (T(gs)) reaching more than 100 °C. The mechanical strength of these ionogels is surprisingly high and even comparable to that of cross-linked protein fibres. Furthermore, the ionogels exhibit rapid self-recovery properties after structural damage caused by deformation. Pseudorotaxanes form from the gelators' benzo[21]crown-7 ethers as the wheels and secondary ammonium ions as the axles despite the competition between that cation and the imidazolium ions of the ionic liquid for crown ether binding. Pseudorotaxane formation as an external chemical stimulus triggers the gel-sol transition of the ionogels.

Fibrous networks with incorporated macrocycles: a chiral stimuli-responsive supramolecular supergelator and its application to biocatalysis in organic media.

A new and versatile, crown ether appended, chiral supergelator has been designed and synthesized based on the bis-urea motif. The introduction of a stereogenic center improved its gelation ability significantly relative to its achiral analogue. This low-molecular-weight gelator forms supramolecular gels in a variety of organic solvents. It is sensitive to multiple chemical stimuli and the sol-gel phase transitions can be reversibly triggered by host-guest interactions. The gel can be used to trap enzymes and release them on demand by chemical stimuli. It stabilizes the microparticles in Pickering emulsions so that enzyme-catalyzed organic reactions can take place in the polar phase inside the microparticles, the organic reactants diffusing through the biphasic interface from the surrounding organic phase. Because of the higher interface area between the organic and polar phases, enzyme activity is enhanced in comparison with simple biphasic systems.