Stable and permeable polyion complex vesicles designed as enzymatic nanoreactors.

Polymeric vesicles are perspective vehicles for fabricating enzymatic nanoreactors towards diverse biomedical and catalytic applications, yet the design of stable and permeable vesicles remains challenging. Herein, we developed polyion complex (PIC) vesicles featuring high stability and a permeable membrane for adequate enzyme loading and activation. Our design relies on co-assembly of an anionic diblock copolymer (PSS96-b-PEO113) with cationic branched poly(ethylenimine) (PEI). The polymer combination endows strong electrostatic interaction between the PSS and PEI building blocks, so their assembly can be implemented at a high salt concentration (500 mM NaCl), under which the charge interaction of the enzyme-polymer is inhibited. This control realizes the successful and safe loading of enzymes associated with the formation of stable PIC vesicles with an intrinsic permeable membrane that is favourable for enhancing enzymatic activity. The control factors for vesicle formation and enzyme loading were investigated, and the general application of loading different enzymes for cascade reaction was validated as well. Our study reveals that proper design and combination of polyelectrolytes is a facile strategy for fabricating stable and permeable polymeric PIC vesicles, which exhibit clear advantages for loading and activating enzymes, consequently boosting their diverse applications as enzymatic nanoreactors.

Switchable Electrostatically Templated Polymerization.

We report a switchable, templated polymerization system where the strength of the templating effect can be modulated by solution pH and/or ionic strength. The responsiveness to these cues is incorporated through a dendritic polyamidoamine-based template of which the charge density depends on pH. The dendrimers act as a template for the polymerization of an oppositely charged monomer, namely sodium styrene sulfonate. We show that the rate of polymerization and maximum achievable monomer conversion are directly related to the charge density of the template, and hence the environmental pH. The polymerization could effectively be switched "ON" and "OFF" on demand, by cycling between acidic and alkaline reaction environments. These findings break ground for a novel concept, namely harnessing co-assembly of a template and growing polymer chains with tunable association strength to create and control coupled polymerization and self-assembly pathways of (charged) macromolecular building blocks.

Template-Free Self-Assembly of Artificial De Novo Viral Coat Proteins into Nanorods: Effects of Sequence, Concentration, and Temperature.

The self-assembly of protein polymers is a promising route to prepare sophisticated functional nanostructures. However, the interplay between protein self-assembly by itself and its co-assembly with a template is not well understood. Silk-based protein polymers that co-assemble with DNA to form rod-like artificial viruses are herein developed and the effects of silk block length, concentration, and temperature in the self-assembly of the proteins alone are characterized by using a combination of bulk dynamic light scattering (DLS) and single-molecule atomic force microscopy (AFM). Protein nanorods were slowly formed (up to hours) through the interaction of the silk-like blocks. The proteins present a silk-length dependent critical elongation concentration, and above it the amount and size of nanorods rapidly increase. Temperature-dependent light scattering data was adequately fitted into a cooperative model of nucleation-elongation. These results are also important to understand the self-assembly of designed viral coat proteins with DNA templates to form artificial virus-like particles and help us to define general guidelines to design proteins with the ability to precisely organize matter at the nanoscale.

Supramolecular Virus-Like Nanorods by Coassembly of a Triblock Polypeptide and Reversible Coordination Polymers.

We investigate a new case of a self-assembly-stimulated self-assembly in which a triblock polypeptide is combined with a anionic coordination polymer of a dipicolinic acid bis-ligand, and d- or f- block metal ions like ZnII or EuIII . The polypeptide not only has a silk-like domain that can fold and stack, but also a C-terminal cationic sequence by which it can interact with the supramolecular (coordination) polyanion. In the presence of all three ingredients (polypeptide, bis-ligand, and metal ions), we observe the initiation and slow growth of well-defined metal-containing nanorods of up to 150 nm in length, proving that self-assembly of the polypeptide is triggered by the self-assembly of the coordination polyelectrolyte and vice versa. The particles, which have a striking resemblance to rod-like viruses, are stable up to 1.2 m NaCl, and can be made fluorescent when lanthanides like EuIII are used, showing the potential to exploit functional properties and applications of virus-like supramolecular structures.

Heparin as a Bundler in a Self-Assembled Fibrous Network of Functionalized Protein-Based Polymers.

Nature shows excellent control over the mechanics of fibrous hydrogels by assembling protein fibers into bundles of well-defined dimensions. Yet, obtaining artificial materials displaying controlled bundling remains a challenge. Here, we developed genetically engineered protein-based polymers functionalized with heparin-binding KRSR domains and show controlled bundling using heparin as a binder. The protein polymer forms fibers upon increasing the pH to physiological values and at higher concentrations fibrous gels. We show that addition of heparin to the protein polymer with incorporated KRSR domains, induces bundling, which results in faster gel formation and stiffer gels. The interactions are expected to be primarily electrostatic and fiber bundling has an optimum when the positive charges of KRSR are approximately in balance with the negative charges of the heparin. Our study suggests that, generally, a straightforward method to control the properties of fibrous gels is to prepare a fiber former with specific binding domains and then simply adding an appropriate amount of binder.

Self-assembly of ultralong polyion nanoladders facilitated by ionic recognition and molecular stiffness.

It is hard to obtain spatially ordered nanostructures via the polyion complexation process due to the inherent flexibility of polymers and isotropicity of ionic interactions. Here we report the formation of polyion assemblies with well-defined, periodically regular internal structure by imparting the proper stiffness to the molecular tile. A stiff bisligand TPE-C4-L2 was designed which is able to form a negatively charged supramolecular polyelectrolyte with transition metal ions. This supramolecular polyelectrolyte slowly self-assembled into polydispersed flat sheets with cocoon-like patterns. Upon the addition of an oppositely charged ordinary polyelectrolyte, the polydispersed cocoons immediately transformed into ultralong, uniform nanoladders as a result of matched ionic density recognition. The supramolecular polyelectrolytes assembled side-by-side, and the negative charges aligned in an array. This structure forced the positively charged polymers to lie along the negative charges so that the perpendicular arrangement of the oppositely charged chains was achieved. Such precise charge recognition will provide insight into the design of advanced materials with hierarchical self-assembled structures.

Rational design of diblock copolymer enables efficient cytosolic protein delivery.

Polymer-mediated cytosolic protein delivery demonstrates a promising strategy for the development of protein therapeutics. Here, we propose a new designed diblock copolymer which realizes efficient cytosolic protein delivery both in vitro and in vivo. The polymer contains one protein-binding block composed of phenylboronic acid (PBA) and N-(3-dimethylaminopropyl) (DMAP) pendant units for protein binding and endosomal escape, respectively, followed by the response to ATP enriched in the cytosol which triggers the protein release. The other block is PEG designed to improve particle size control and circulation in vivo. By optimizing the block composition, sequence and length of the copolymer, the optimal one (BP20) was identified with the binding block containing 20 units of both PBA and DMAP, randomly distributed along the chain. When mixed with proteins, the BP20 forms stable nanoparticles and mediates efficient cytosolic delivery of a wide range of proteins including enzymes, toxic proteins and CRISPR/Cas9 ribonucleoproteins (RNP), to various cell lines. The PEG block, especially when further modified with folic acid (FA), enables tumor-targeted delivery of Saporin in vivo, which significantly suppresses the tumor growth. Our results shall inspire the design of novel polymeric vehicles with robust capability for cytosolic protein delivery, which holds great potential for both biological research and therapeutic applications.

Covalently attached organic monolayers onto silicon carbide from 1-alkynes: molecular structure and tribological properties.

In order to achieve improved tribological and wear properties at semiconductor interfaces, we have investigated the thermal grafting of both alkylated and fluorine-containing ((C(x)F(2x+1))-(CH2)n-) 1-alkynes and 1-alkenes onto silicon carbide (SiC). The resulting monolayers display static water contact angles up to 120°. The chemical composition of the covalently bound monolayers was studied by X-ray photoelectron spectroscopy (XPS), infrared reflection-absorption spectroscopy (IRRAS), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. These techniques indicate the presence of acetal groups at the organic-inorganic interface of alkyne-modified SiC surfaces. The tribological properties of the resulting organic monolayers with fluorinated or nonfluorinated end groups were explored using atomic force microscopy (AFM). It was found that the fluorinated monolayers exhibit a significant reduction of adhesion forces, friction forces, and wear resistance compared with non-fluorinated molecular coatings and especially bare SiC substrates. The successful combination of hydrophobicity and excellent tribological properties makes these strongly bound, fluorinated monolayers promising candidates for application as a thin film coating in high-performance microelectronic devices.

Physical gels based on charge-driven bridging of nanoparticles by triblock copolymers.

We have prepared an aqueous physical gel consisting of negatively charged silica nanoparticles bridged by ABA triblock copolymers, in which the A blocks are positively charged and the B block is neutral and water-soluble. Irreversible aggregation of the silica nanoparticles was prevented by precoating them with a neutral hydrophilic polymer. Both the elastic plateau modulus and the relaxation time increase slowly as the gel ages, indicating an increase both in the number of active bridges and in the strength with which the end blocks are adsorbed. The rate of this aging process can be increased significantly by applying a small shear stress to the sample. Our results indicate that charge-driven bridging of nanoparticles by triblock copolymers is a promising strategy for thickening of aqueous particle containing materials, such as water-based coatings.

Emerging applications of stimuli-responsive polymer materials.

Responsive polymer materials can adapt to surrounding environments, regulate transport of ions and molecules, change wettability and adhesion of different species on external stimuli, or convert chemical and biochemical signals into optical, electrical, thermal and mechanical signals, and vice versa. These materials are playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering and 'smart' optical systems, as well as biosensors, microelectromechanical systems, coatings and textiles. We review recent advances and challenges in the developments towards applications of stimuli-responsive polymeric materials that are self-assembled from nanostructured building blocks. We also provide a critical outline of emerging developments.

Relaxation dynamics at different time scales in electrostatic complexes: time-salt superposition.

In this Letter we show that in the rheology of electrostatically assembled soft materials, salt concentration plays a similar role as temperature for polymer melts, and as strain rate for soft solids. We rescale linear and nonlinear rheological data of a set of model electrostatic complexes at different salt concentrations to access a range of time scales that is otherwise inaccessible. This provides new insights into the relaxation mechanisms of electrostatic complexes, which we rationalize in terms of a microscopic mechanism underlying salt-enhanced activated processes.

Nanowires formed by the co-assembly of a negatively charged low-molecular weight gelator and a zwitterionic polythiophene.

Conjugated organic nanowires have been prepared by co-assembling a carboxylate containing low-molecular weight gelator (LMWG) and an amino acid substituted polythiophene derivative (PTT). Upon introducing the zwitterionic polyelectrolyte PTT to a basic molecular solution of the organogelator, the negative charges on the LMWG are compensated by the positive charges of the PTT. As a result, nanowires form through co-assembly. These nanowires are visualized by both transmission electron microscopy (TEM) and atomic force microscopy (AFM). Depending on the concentration and ratio of the components these nanowires can be micrometers long. These measurements further suggest that the aggregates adopt a helical conformation. The morphology of these nanowires are studied with fluorescent confocal laser scanning microscopy (CLSM). The interactions between LMWG and PTT are characterized by steady-state and time-resolved fluorescence spectroscopy studies. The steady-state spectra indicate that the backbone of the PTT adopts a more planar and more aggregated conformation when interacting with LMWG. The time- resolved fluorescence decay studies confirm this interpretation.

Binding of beta-lactoglobulin to pectins varying in their overall and local charge density.

The formation of complexes between proteins and polysaccharides is of great importance for many food systems like foams, emulsions, acidified milk drinks, and so on. The complex formation between beta-lactoglobulin (beta-lg) and pectins with a well-defined physicochemical fine structure has been studied to elucidate the influence of overall charge and local charge density of pectin on the complex formation. Binding isotherms of beta-lg to pectin are constructed using fluorescence anisotropy, which is shown to be an excellent technique for this purpose, as it is fast and requires low sample volumes. From the binding isotherms the maximal adsorbed amount, binding constant (k(obs)) and the cooperativity of binding are obtained at different ionic strengths. The Hill model is used to fit the binding isotherms and is shown to be preferable over a Langmuir fit. At pH 4.25, k(obs) shows a maximum at an ionic strength of 10 mM when using a low methyl esterified pectin (LMP) due to the balance of attractive and repulsive electrostatic forces between beta-lg and pectin and beta-lg neighbors. For two high methyl esterified pectins, one with a blockwise distribution of methyl esters (HMP(B)) and one with a random distribution (HMP(R)), this ionic strength maximum is absent and k(obs) decreases with increasing ionic strength. k(obs) is found to be largest for LMP and HMP(B) and considerably lower for HMP(R). A positive cooperativity is observed for both LMP (above an ionic strength of 45 mM) and HMP(R) (above an ionic strength of 15 mM) but not for HMP(B). Positive cooperativity is thought to be caused by a rearrangement of the pectin helix structure caused by binding of beta-lg, thus creating new or binding sites with a higher affinity. To attain strong binding of beta-lg to pectin it is preferable to use a pectin with a blockwise distribution of methyl esters. When complex formation takes place in high ionic strength media an LMP gives the best results, while at low ionic strength a high methyl esterified pectin with blockwise distribution may give better results, due to reduced electrostatic repulsion between both pectin and beta-lg and beta-lg neighbors.

Intermittent dynamics in transient polymer networks under shear: signs of self-organized criticality.

In this paper we demonstrate an unusual behavior in the shear-banded flow of a viscoelastic fluid. We report large and patterned fluctuations in the shear stress in an apparently fluid material undergoing steady shear, which we interpret as an intermittent and microscopic fracture and self-healing process. The statistical pattern of the fluctuations is indicative of self-organized criticality, and their magnitude can be directly related to the constitutive instability that underlies the shear banding.

Temperature responsive complex coacervate core micelles with a PEO and PNIPAAm corona.

In aqueous solutions at room temperature, poly( N-methyl-2-vinyl pyridinium iodide)- block-poly(ethylene oxide), P2MVP 38- b-PEO 211 and poly(acrylic acid)- block-poly(isopropyl acrylamide), PAA 55- b-PNIPAAm 88 spontaneously coassemble into micelles, consisting of a mixed P2MVP/PAA polyelectrolyte core and a PEO/PNIPAAm corona. These so-called complex coacervate core micelles (C3Ms), also known as polyion complex (PIC) micelles, block ionomer complexes (BIC), and interpolyelectrolyte complexes (IPEC), respond to changes in solution pH and ionic strength as their micellization is electrostatically driven. Furthermore, the PNIPAAm segments ensure temperature responsiveness as they exhibit lower critical solution temperature (LCST) behavior. Light scattering, two-dimensional 1H NMR nuclear Overhauser effect spectrometry, and cryogenic transmission electron microscopy experiments were carried out to investigate micellar structure and solution behavior at 1 mM NaNO 3, T = 25, and 60 degrees C, that is, below and above the LCST of approximately 32 degrees C. At T = 25 degrees C, C3Ms were observed for 7 < pH < 12 and NaNO 3 concentrations below approximately 105 mM. The PEO and PNIPAAm chains appear to be (randomly) mixed within the micellar corona. At T = 60 degrees C, onion-like complexes are formed, consisting of a PNIPAAm inner core, a mixed P2MVP/PAA complex coacervate shell, and a PEO corona.

Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP-HPLC and UV-detection.

To obtain insight in mechanisms of action of extracellular adenosine triphosphate (ATP) and adenosine, a simple HPLC method has been optimized and applied to investigate ATP metabolism in human whole blood ex vivo. This method provided good chromatographic resolution and peak shape for all eight compounds within a 19 min run time. The baseline was clean, the lower limit of quantification was below 0.3 micromol/L for all adenine nucleotides and the method demonstrated good linearity. Within-day precision ranged from 0.7 to 5.9% and between-days from 2.6 to 15.3%. Simplicity and simultaneous detection of ATP and its metabolites make this method suitable for clinical pharmacokinetic studies.

Fat retention at the tongue and the role of saliva: adhesion and spreading of 'protein-poor' versus 'protein-rich' emulsions.

Fat perception of food emulsions has been found to relate to in-mouth friction. Previously, we have shown that friction under mouth-like conditions strongly depends on the sensitivity of protein-stabilized emulsion droplets to coalescence. Here, we investigated whether this also implies that oral fat retention depends in a similar manner on the stability of the emulsion droplets against coalescence. We investigate the separate contributions of droplet adhesion and droplet spreading to fat retention at the tongue, as well as the role of saliva. We perform ex vivo (Confocal Raman Spectroscopy; Confocal Scanning Laser Microscopy) experiments using pig's tongue surfaces in combination with human in vivo experiments. These reveal that protein-poor (unstable) emulsions are retained more at the tongue than protein-rich (stable) emulsions. Furthermore, the layer formed by adhering protein-poor droplets is more stable against rinsing. Saliva is found to be very efficient in removing fat and emulsion droplets from the oral surface but its role in fat retention needs further research. We relate our results to the colloidal forces governing droplet adhesion and spreading.

Electrostatic stiffening and induced persistence length for coassembled molecular bottlebrushes.

A self-consistent field analysis for tunable contributions to the persistence length of isolated semiflexible polymer chains including electrostatically driven coassembled deoxyribonucleic acid (DNA) bottlebrushes is presented. When a chain is charged, i.e., for polyelectrolytes, there is, in addition to an intrinsic rigidity, an electrostatic stiffening effect, because the electric double layer resists bending. For molecular bottlebrushes, there is an induced contribution due to the grafts. We explore cases beyond the classical phantom main-chain approximation and elaborate molecularly more realistic models where the backbone has a finite volume, which is necessary for treating coassembled bottlebrushes. We find that the way in which the linear charge density or the grafting density is regulated is important. Typically, the stiffening effect is reduced when there is freedom for these quantities to adapt to the curvature stresses. Electrostatically driven coassembled bottlebrushes, however, are relatively stiff because the chains have a low tendency to escape from the compressed regions and the electrostatic binding force is largest in the convex part. For coassembled bottlebrushes, the induced persistence length is a nonmonotonic function of the polymer concentration: For low polymer concentrations, the stiffening grows quadratically with coverage; for semidilute polymer concentrations, the brush chains retract and regain their Gaussian size. When doing so, they lose their induced persistence length contribution. Our results correlate well with observed physical characteristics of electrostatically driven coassembled DNA-bioengineered protein-polymer bottlebrushes.

Characteristic differences in the formation of complex coacervate core micelles from neodymium and zinc-based coordination polymers.

In this paper we compare the formation of complex coacervate core micelles (C3Ms) from two different tricompontent mixtures, namely neodymium, the bisligand L2EO4 and the poly(cation)-block-poly(neutral) diblock copolymer P2MVP41-b-PEO205, and zinc, L2EO4 and P2MVP41-b-PEO205 mixed systems. Three sets of titration experiments were carried out for each system: (i) titration of diblock copolymer P2MVP41-b-PEO205 with the stoichiometric mixture of metal ions and bisligands, (ii) titration of a mixture of diblock copolymer and bisligand with metal ions, and (iii) titration of a mixture of diblock copolymer and metal ions with bisligands. In all the above three cases, micelles are found to form either in a broad range of charge ratios or in a broad range of metal/bisligand ratios. Upon addition of Nd2-(L2EO4)3 coordination polymer to P2MVP41-b-PEO205 solution, and upon addition of Nd3+ to a mixture of L2EO4 and P2MVP41-b-PEO205, micelles are found to form immediately after the first addition, whereas micelles show up in the similar zinc system only after a certain threshold Zn-(L2EO4) or Zn2+ concentration. This difference can be traced to the different structures of the Nd2-(L2EO4)3 and Zn-(L2EO4) coordination compounds. At very low concentrations, Zn-(L2EO4) are ring-like oligomers, but Nd2-(L2EO4)3 are larger networks. The network structure favors the formation of coacervate micellar core with P2MVP41-b-PEO205. Moreover, excess of Nd3+ ions will break up the C3Ms, while the same amount of Zn2+ has hardly any effect on the C3Ms. The breakdown of C3Ms by Nd3+ is due to the charge inversion of the coordination complex with increasing [Nd3+]/[L2EO4] ratio, which results in repulsive interaction between the coordination complex and the diblock copolymer, whereas no such interaction can occur in the zinc system.

Wormlike aggregates from a supramolecular coordination polymer and a diblock copolymer.

The formation of wormlike micelles in mixed systems of a supramolecular coordination polymer Zn-L2EO4 and a diblock copolymer P2MVP41-b-PEO205 is investigated by light scattering and Cryo-TEM. By direct mixing at a stoichiometric charge ratio, the above mixtures proved to be capable of formation of spherical micelles with a radius of about 25 nm (Yan et al. Angew. Chem., Int. Ed.; 2007, 46, 1807-1809). Lately, we find wormlike micelles with a hydrodynamic radius >150 nm in a mixture with excess positive charge, that is, a negative charge fraction f- < 0.5. The transformation between wormlike and spherical micelles can be realized by variation of the mixing ratio through different protocols. Upon addition of negatively charged Zn-L2EO4 to a mixture with excess positively charged P2MVP41-b-PEO205, most of the wormlike micelles are transformed into spherical ones; upon addition of positively charged P2MVP41-b-PEO205 to a mixture of pure spherical micelles, wormlike micelles can be produced again. The effect of sample preparation protocol, sample history, and concentration on this transformation process is systematically reported in this article. A possible mechanism for the formation of wormlike micelles is proposed.