Polyelectrolyte-protein synergism: pH-responsive polyelectrolyte/insulin complexes as versatile carriers for targeted protein and drug delivery.

The co-assembly of polyelectrolytes (PE) with proteins offers a promising approach for designing complex structures with customizable morphologies, charge distribution, and stability for targeted cargo delivery. However, the complexity of protein structure limits our ability to predict the properties of the formed nanoparticles, and our goal is to identify the key triggers of the morphological transition in protein/PE complexes and evaluate their ability to encapsulate multivalent ionic drugs. A positively charged PE can assemble with a protein at pH above isoelectric point due to the electrostatic attraction and disassemble at pH below isoelectric point due to the repulsion. The additional hydrophilic block of the polymer should stabilize the particles in solution and enable them to encapsulate a negatively charged drug in the presence of PE excess. We demonstrated that diblock copolymers, poly(ethylene oxide)-block-poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide)-block-poly(N,N,N-trimethylammonioethyl methacrylate), consisting of a polycation block and a neutral hydrophilic block, reversibly co-assemble with insulin in pH range between 5 and 8. Using small-angle neutron and X-ray scattering (SANS, SAXS), we showed that insulin arrangement within formed particles is controlled by intermolecular electrostatic forces between protein molecules, and can be tuned by varying ionic strength. For the first time, we observed by fluorescence that formed protein/PE complexes with excess of positive charges exhibited potential for encapsulating and controlled release of negatively charged bivalent drugs, protoporphyrin-IX and zinc(II) protoporphyrin-IX, enabling the development of nanocarriers for combination therapies with adjustable charge, stability, internal structure, and size.

Advances in Small Angle Neutron Scattering on Polysaccharide Materials.

Polysaccharide materials and biomaterials gain the focus of intense research owing to their great versatility in chemical structures and modification possibilities, as well as their biocompatibility, degradability, and sustainability features. This review focuses on the recent advances in the application of SANS on polysaccharide systems covering a broad range of materials such as nanoparticulate assemblies, hydrogels, nanocomposites, and plant-originating nanostructured systems. It motivates the use of SANS in its full potential by demonstrating the features of contrast variation and contrast matching methods and by reporting the methodologies for data analysis and interpretation. As these soft matter systems may be organized in multiple length scales depending on the interactions and chemical bonds between their components, SANS offers exceptional and unique opportunities for advanced characterization and optimization of new nanostructured polysaccharide materials.

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.

Simultaneous SANS/FTIR measurement system incorporating the ATR sampling method.

Small-angle neutron scattering (SANS) is widely used as a powerful technique to study the higher-order structure of soft matter. To increase the reliability of SANS profile analysis for complex multi-component systems, combining different types of structural information obtained by other methods is desirable. A simultaneous measurement system combining SANS and Fourier transform infrared (FTIR) spectroscopy meets this objective. It is beneficial for targets where matching the timing of structural changes between experiments is difficult, but the issue is that samples suitable for SANS are too thick for the typical transmission FTIR method. To overcome this problem, a new simultaneous measurement system that employs the attenuated total reflectance (ART) sampling method for FTIR spectroscopy has been developed.

Interchain Hydrodynamic Interaction and Internal Friction of Polyelectrolytes.

Polyelectrolytes (PE) are polymeric macromolecules in aqueous solutions characterized by their chain topology and intrinsic charge in a neutralizing fluid. Structure and dynamics are related to several characteristic screening length scales determined by electrostatic, excluded volume, and hydrodynamic interactions. We examine PE dynamics in dilute to semidilute conditions using dynamic light scattering, neutron spinecho spectroscopy, and pulse field gradient NMR spectroscopy. We connect macroscopic diffusion to segmental chain dynamics, revealing a decoupling of local chain dynamics from interchain interactions. Collective diffusion is described within a colloidal picture, including electrostatic and hydrodynamic interactions. Chain dynamics is characterized by the classical Zimm model of a neutral chain retarded by internal friction. We observe that hydrodynamic interactions are not fully screened between chains and that the internal friction within the chain increases with an increase in ion condensation on the chain.

Extended Q-range small-angle neutron scattering to understand the morphology of proton-exchange membranes: the case of the functionalized syndiotactic-polystyrene model system.

Semi-crystalline polymers exhibit microphase separation into crystalline and amorphous domains characterized by multiple structural levels with sizes ranging from ångströms to hundreds of nanometres. The combination of small-angle (SANS) and wide-angle (WANS) neutron scattering on the same beamline enables reliable in situ characterization of such materials under application-relevant conditions, with the unique advantage of contrast variation by controlled labelling, allowing the structure of such multi-component systems to be resolved in detail. This paper reports a structural analysis performed on deuterated polymer membranes based on syndiotactic polystyrene (sPS) using an extended Q-range SANS and WANS combination, always with the same neutron scattering instrument, either a pinhole SANS diffractometer installed at a research reactor or a 'small- and wide-angle' time-of-flight diffractometer installed at a neutron spallation source. sPS is a semi-crystalline material that becomes hydrophilic and proton conducting when suitable functionalization is achieved by thin film sulfonation, and can form various co-crystalline complexes (clathrates) with small organic molecules stored in the crystalline phase as guests in the vacancies between the polymer helices. Therefore, this material is interesting not only for its conducting properties but also for its versatility as a model system to evaluate the usefulness of extended Q-range neutron scattering in such studies. Variation of neutron contrast was achieved in the amorphous hydrophilic phase by using H2O or D2O to hydrate the membranes and in the crystalline phase by loading the clathrates with deuterated or protonated guest molecules. The experimental approach, the advantages and limitations of the two types of instrumentation used in such analyses, and the main results obtained with respect to the structural characterization of sulfonated sPS membranes under different hydration and temperature conditions are reported, and the potential of this method for similar structural studies on other semi-crystalline polymeric materials is discussed.

Stability of Nanopeptides: Structure and Molecular Exchange of Self-assembled Peptide Fibers.

Often nanostructures formed by self-assembly of small molecules based on hydrophobic interactions are rather unstable, causing morphological changes or even dissolution when exposed to changes in aqueous media. In contrast, peptides offer precise control of the nanostructure through a range of molecular interactions where physical stability can be engineered in and, to a certain extent, decoupled from size via rational design. Here, we investigate a family of peptides that form beta-sheet nanofibers and demonstrate a remarkable physical stability even after attachment of poly(ethylene glycol). We employed small-angle neutron/X-ray scattering, circular dichroism spectroscopy, and molecular dynamics simulation techniques to investigate the detailed nanostructure, stability, and molecular exchange. The results for the most stable sequence did not reveal any structural alterations or unimer exchange for temperatures up to 85 °C in the biologically relevant pH range. Only under severe mechanical perturbation (i.e., tip sonication) would the fibers break up, which is reflected in a very high activation barrier for unimer exchange of ∼320 kJ/mol extracted from simulations. The results give important insight into the relation between molecular structure and stability of peptide nanostructure that is important for, e.g., biomedical applications.

Block Copolymer Adsorption on the Surface of Multi-Walled Carbon Nanotubes for Dispersion in N,N Dimethyl Formamide.

This research aims to characterize the adsorption morphology of block copolymer dispersants of the styrene-block-4-vinylpyridine family (S4VP) on the surface of multi-walled carbon nanotubes (MWCNT) in a polar organic solvent, N,N-dimethyl formamide (DMF). Good, unagglomerated dispersion is important in several applications such as fabricating CNT nanocomposites in a polymer film for electronic or optical devices. Small-angle neutron scattering (SANS) measurements, using the contrast variation (CV) method, are used to evaluate the density and extension of the polymer chains adsorbed on the nanotube surface, which can yield insight into the means of successful dispersion. The results show that the block copolymers adsorb onto the MWCNT surface as a continuous coverage of low polymer concentration. Poly(styrene) (PS) blocks adsorb more tightly, forming a 20 Å layer containing about 6 wt.% PS, whereas poly(4-vinylpyridine) (P4VP) blocks emanate into the solvent, forming a thicker shell (totaling 110 Å in radius) but of very dilute (<1 wt.%) polymer concentration. This indicates strong chain extension. Increasing the PS molecular weight increases the thickness of the adsorbed layer but decreases the overall polymer concentration within it. These results are relevant for the ability of dispersed CNTs to form a strong interface with matrix polymers in composites, due to the extension of the 4VP chains allowing for entanglement with matrix chains. The sparse polymer coverage of the CNT surface may provide sufficient space to form CNT-CNT contacts in processed films and composites, which are important for electrical or thermal conductivity.

Microscopic Determination of the Local Hydration Number of Polymer Electrolyte Membranes Using SANS Partial Scattering Function Analysis.

The conventional hydration number, λ, of a polymer electrolyte membrane (PEM) is estimated by the gravimetric measurement of the total water uptake in the membrane. This is the overall water molecules divided by the number of ionic groups covering the water distribution from the macroscale to the molecular level. For a more precise evaluation of the local ion-water interactions and ion transport efficiency in hydrophilic channels, in contrast to λ, we here propose an index, local hydration number (λlocal), defined as the number of water molecules located adjacent to and interacting with ionic groups in a PEM, per ionic group in the hydrophilic channels. We introduce an experimental method using partial scattering function analysis through a contrast-variation small-angle neutron scattering technique to precisely determine λlocal at the micro- (nano-) scale to the molecular level for PEMs. When applied to the benchmark material Nafion and the well-studied radiation-grafted PEM, consisting of poly(styrene sulfonic acid) grafted onto poly(ethylene-co-tetrafluoroethylene) (ETFE-g-PSSA), the obtained λlocal was close to the conventional λ. This means all water molecules interacted with sulfonic acid groups, which is much different from the case of anion-type grafted PEMs. Comparing the λlocal value obtained in this study with the conventional λ value confirmed that water existed only in nanostructured hydrophilic channels in these PEMs. The result of this work provides a route to understand local molecular structures of high-performing PEMs for energy conversion applications.

Protein-induced transformation of unilamellar to multilamellar vesicles triggered by a polysaccharide.

We report on the morphological transitions of didodecyldimethylammonium bromide (DDAB) cationic vesicles and hybrid DDAB/hyaluronic acid (HA) vesicles upon addition of BSA at pH 7 where BSA is overall negatively charged. Small angle neutron scattering (SANS) is used to extract the size distributions of the nanovesicles, the thickness of the DDAB bilayers and their lamellarity. Although the HA-decorated DDAB vesicles contain the negatively charged polysaccharide the interaction with BSA appears to be more intense in comparison to bare vesicles. Characteristic peaks in the SANS patterns indicate the presence of multilamellar interfaces while the formation of multilamellar vesicles induced by BSA depends on the amount of added HA. Consequently, higher lamellarities are observed at higher BSA contents. This work demonstrates a simple methodology to tune the encapsulation of globular proteins in vesicular nanoassemblies by affecting their lamellarity and has direct implications on the application of vesicles and liposomes in protein delivery.

Small-Angle Neutron Scattering Reveals the Nanostructure of Liposomes with Embedded OprF Porins of Pseudomonas aeruginosa.

The use of liposomes as drug delivery systems emerged in the last decades in view of their capacity and versatility to deliver a variety of therapeutic agents. By means of small-angle neutron scattering (SANS), we performed a detailed characterization of liposomes containing outer membrane protein F (OprF), the main porin of the Pseudomonas aeruginosa bacterium outer membrane. These OprF-liposomes are the basis of a novel vaccine against this antibiotic-resistant bacterium, which is one of the main hospital-acquired pathogens and causes each year a significant number of deaths. SANS data were analyzed by a specific model we created to quantify the crucial information about the structure of the liposome containing OprF, including the lipid bilayer structure, the amount of protein in the lipid bilayer, the average protein localization, and the effect of the protein incorporation on the lipid bilayer. Quantification of such structural information is important to enhance the design of liposomal delivery systems for therapeutic applications.

Maleimide-Decorated PEGylated Mucoadhesive Liposomes for Ocular Drug Delivery.

Liposomes are promising spherical vesicles for topical drug delivery to the eye. Several types of vesicles were formulated in this study, including conventional, PEGylated, and maleimide-decorated PEGylated liposomes. The physicochemical characteristics of these liposomes, including their size, zeta potential, ciprofloxacin encapsulation efficiency, loading capacity, and release, were evaluated. The structure of these liposomes was examined using dynamic light scattering, transmission electron microscopy, and small angle neutron scattering. The ex vivo corneal and conjunctival retention of these liposomes were examined using the fluorescence flow-through method. Maleimide-decorated liposomes exhibited the best retention performance on bovine conjunctiva compared to other types of liposomes studied. Poor retention of all liposomal formulations was observed on bovine cornea.

Quantifying the Solution Structure of Metal Nanoclusters Using Small-Angle Neutron Scattering.

Metal nanoclusters are a unique class of synthetic material, as their crystal structures can be resolved using X-ray diffraction, and their chemical formula can be precisely determinated from mass spectroscopy. However, a complete structure characterization by these two techniques is often a challenging task. Here, we utilize small-angle neutron scattering (SANS) to directly quantify the key structure parameters of a series of silver and gold nanoclusters in solution. The results not only correlate well to their crystallographic structures, but also allow the quantification of the counterions layer surrounding charged nanoclusters in solution. Furthermore, when combining with X-ray scattering, it is possible to estimate the molecular weight of both the metal core and the ligand shell of nanoclusters. This work offers an alternative characterization tool for nanoclusters without the requirement of crystallization or gas phase ionization.

Variation of Structural and Dynamical Flexibility of Myelin Basic Protein in Response to Guanidinium Chloride.

Myelin basic protein (MBP) is intrinsically disordered in solution and is considered as a conformationally flexible biomacromolecule. Here, we present a study on perturbation of MBP structure and dynamics by the denaturant guanidinium chloride (GndCl) using small-angle scattering and neutron spin-echo spectroscopy (NSE). A concentration of 0.2 M GndCl causes charge screening in MBP resulting in a compact, but still disordered protein conformation, while GndCl concentrations above 1 M lead to structural expansion and swelling of MBP. NSE data of MBP were analyzed using the Zimm model with internal friction (ZIF) and normal mode (NM) analysis. A significant contribution of internal friction was found in compact states of MBP that approaches a non-vanishing internal friction relaxation time of approximately 40 ns at high GndCl concentrations. NM analysis demonstrates that the relaxation rates of internal modes of MBP remain unaffected by GndCl, while structural expansion due to GndCl results in increased amplitudes of internal motions. Within the model of the Brownian oscillator our observations can be rationalized by a loss of friction within the protein due to structural expansion. Our study highlights the intimate coupling of structural and dynamical plasticity of MBP, and its fundamental difference to the behavior of ideal polymers in solution.

On the Proton Conduction Pathways in Polyelectrolyte Membranes Based on Syndiotactic-Polystyrene.

When functionalized by the solid-state sulfonation process, the amorphous regions of the semi-crystalline syndiotactic-polystyrene (sPS) become hydrophilic, and thus can conduct protons upon membrane hydration, which increases the interest in this material as a potential candidate for applications with proton exchange membranes. The resistance of sulfonated sPS to oxidative decomposition can be improved by doping the membrane with fullerenes. In previous work, we have described the morphology in hydrated sulfonated sPS films doped with fullerenes on different length scales as determined by small-angle neutron scattering (SANS) and the structural changes in such membranes as a function of the degree of hydration and temperature. In the current work, we report on the relationship between the morphology of hydrated domains as obtained by SANS and the proton conductivity in sulfonated sPS-fullerene composite membranes at different temperature and relative humidity (RH) conditions. Based on this combined experimental approach, clear evidence for the formation and evolution of the hydrated domains in functionalized sPS membranes has been provided and a better understanding of the hydration and conductivity pathways in this material has been obtained.

Influence of Chain Length of Gradient and Block Copoly(2-oxazoline)s on Self-Assembly and Drug Encapsulation.

Amphiphilic gradient copolymers represent a promising alternative to extensively used block copolymers due to their facile one-step synthesis by statistical copolymerization of monomers of different reactivity. Herein, an in-depth analysis is provided of micelles based on amphiphilic gradient poly(2-oxazoline)s with different chain lengths to evaluate their potential for micellar drug delivery systems and compare them to the analogous diblock copolymer micelles. Size, morphology, and stability of self-assembled nanoparticles, loading of hydrophobic drug curcumin, as well as cytotoxicities of the prepared nanoformulations are examined using copoly(2-oxazoline)s with varying chain lengths and comonomer ratios. In addition to several interesting differences between the two copolymer architecture classes, such as more compact self-assembled structures with faster exchange dynamics for the gradient copolymers, it is concluded that gradient copolymers provide stable curcumin nanoformulations with comparable drug loadings to block copolymer systems and benefit from more straightforward copolymer synthesis. The study demonstrates the potential of amphiphilic gradient copolymers as a versatile platform for the synthesis of new polymer therapeutics.

Evidence of supercoolable nanoscale water clusters in an amorphous ionic liquid matrix.

Nanoscale water clusters in an ionic liquid matrix, also called "water pockets," were previously found in some mixtures of water with ionic liquids containing hydrophilic anions. However, in these systems, at least partial crystallization occurs upon supercooling. In this work, we show for mixtures of 1-butyl-3-methylimidazolium dicyanamide with water that none of the components crystallizes up to a water content of 72 mol. %. The dynamics of the ionic liquid matrix is monitored from above room temperature down to the glass transition by combining depolarized dynamic light scattering with broadband dielectric and nuclear magnetic resonance spectroscopy, revealing that the matrix behaves like a common glass former and stays amorphous in the whole temperature range. Moreover, we demonstrate by a combination of Raman spectroscopy, small angle neutron scattering, and molecular dynamics simulation that, indeed, nanoscale water clusters exist in this mixture.

Separation of the inelastic and elastic scattering in time-of-flight mode on the pinhole small-angle neutron scattering diffractometer K-WS-2.

To study and control the incoherent inelastic background in small-angle neutron scattering, which makes a significant contribution to the detected scattering from hydrocarbon systems, the KWS-2 small-angle neutron scattering diffractometer operated by the Jülich Centre for Neutron Science (JCNS) at Heinz-Maier Leibnitz Zentrum (MLZ), Garching, Germany, was equipped with a secondary single-disc chopper that is placed in front of the sample stage. This makes it possible to record in time-of-flight mode the scattered neutrons in the high-Q regime of the instrument (i.e. short incoming wavelengths and detection distances) and to discard the inelastic component from the measured data. Examples of measurements on different materials routinely used as standard samples, sample containers and solvents in the experiments at KWS-2 are presented. When only the elastic region of the spectrum is used in the data-reduction procedure, a decrease of up to two times in the incoherent background of the experimentally measured scattering cross section may be obtained. The proof of principle is demonstrated on a solution of bovine serum albumin in D2O.

Structural Organization of Cardiolipin-Containing Vesicles as Models of the Bacterial Cytoplasmic Membrane.

The bacterial cytoplasmic membrane is the innermost bacterial membrane and is mainly composed of three different phospholipid species, i.e., phosphoethanolamine (PE), phosphoglycerol (PG), and cardiolipin (CL). In particular, PG and CL are responsible for the negative charge of the membrane and are often the targets of cationic antimicrobial agents. The growing resistance of bacteria toward the available antibiotics requires the development of new and more efficient antibacterial drugs. In this context, studying the physicochemical properties of the bacterial cytoplasmic membrane is pivotal for understanding drug-membrane interactions at the molecular level as well as for designing drug-testing platforms. Here, we discuss the preparation and characterization of PE/PG/CL vesicle suspensions, which contain all of the main lipid components of the bacterial cytoplasmic membrane. The vesicle suspensions were characterized by means of small-angle neutron scattering, dynamic light scattering, and electron paramagnetic spectroscopy. By combining solution scattering and spectroscopy techniques, we propose a detailed description of the impact of different CL concentrations on the structure and dynamics of the PE/PG bilayer. CL induces the formation of thicker bilayers, which exhibit higher curvature and are overall more fluid. The experimental results contribute to shed light on the structure and dynamics of relevant model systems of the bacterial cytoplasmic membrane.

Chain-End Effects on Supramolecular Poly(ethylene glycol) Polymers.

In this work we present a fundamental analysis based on small-angle scattering, linear rheology and differential scanning calorimetry (DSC) experiments of the role of different hydrogen bonding (H-bonding) types on the structure and dynamics of chain-end modified poly(ethylene glycol) (PEG) in bulk. As such bifunctional PEG with a molar mass below the entanglement mass Me is symmetrically end-functionalized with three different hydrogen bonding (H-bonding) groups: thymine-1-acetic acid (thy), diamino-triazine (dat) and 2-ureido-4[1H]-pyrimidinone (upy). A linear block copolymer structure and a Newtonian-like dynamics is observed for PEG-thy/dat while results for PEG-upy structure and dynamics reveal a sphere and a network-like behavior, respectively. These observations are concomitant with an increase of the Flory-Huggins interaction parameter from PEG-thy/dat to PEG-upy that is used to quantify the difference between the H-bonding types. The upy association into spherical clusters is established by the Percus-Yevick approximation that models the inter-particle structure factor for PEG-upy. Moreover, the viscosity study reveals for PEG-upy a shear thickening behavior interpreted in terms of the free path model and related to the time for PEG-upy to dissociate from the upy clusters, seen as virtual crosslinks of the formed network. Moreover, a second relaxation time of different nature is also obtained from the complex shear modulus measurements of PEG-upy by the inverse of the angular frequency where G' and G'' crosses from the network-like to glass-like transition relaxation time, which is related to the segmental friction of PEG-upy polymeric network strands. In fact, not only do PEG-thy/dat and PEG-upy have different viscoelastic properties, but the relaxation times found for PEG-upy are much slower than the ones for PEG-thy/dat. However, the activation energy related to the association dynamics is very similar for both PEG-thy/dat and PEG-upy. Concerning the segmental dynamics, the glass transition temperature obtained from both rheological and calorimetric analysis is similar and increases for PEG-upy while for PEG-thy/dat is almost independent of association behavior. Our results show how supramolecular PEG properties vary by modifying the H-bonding association type and changing the molecular Flory-Huggins interaction parameter, which can be further explored for possible applications.