Dynamics of individual inkjet printed picoliter droplet elucidated by high speed laser speckle imaging.

Inkjet printing is a ubiquitous consumer and industrial process that involves concomitant processes of droplet impact, wetting, evaporation, and imbibement into a substrate as well as consequential substrate rearrangements and remodeling. In this work, we perform a study on the interaction between ink dispersions of different composition on substrates of increasing complexity to disentangle the motion of the liquid from the dynamic response of the substrate. We print three variations of pigmented inks and follow the ensuing dynamics at millisecond and micron time and length scales until complete drying using a multiple scattering technique, laser speckle imaging (LSI). Measurements of the photon transport mean free path, l*, for the printed inks and substrates show that the spatial region of information capture is the entire droplet volume and a depth within the substrate of a few µm beneath the droplet. Within this spatial confinement, LSI is an ideal approach for studying the solid-liquid transition at these small length and time scales by obtaining valid g2 and d2 autocorrelation functions and interpreting these dynamic changes under through kymographs. Our in situ LSI results show that droplets undergo delamination and cracking processes arising during droplet drying, which are confirmed by post mortem SEM imaging.

LCST polymers with UCST behavior.

In this study, temperature dependent behavior of dense dispersions of core crosslinked flower-like micelles is investigated. Micelles were prepared by mixing aqueous solutions of two ABA block copolymers with PEG B-blocks and thermosensitive A-blocks containing PNIPAM and crosslinkable moieties. At a temperature above the lower critical solution temperature (LCST), self-assembly of the polymers resulted in the formation of flower-like micelles with a hydrophilic PEG shell and a hydrophobic core. The micellar core was stabilized by native chemical ligation (NCL). Above the LCST, micelles displayed a radius of ∼35 nm, while a radius of ∼48 nm was found below the LCST due to hydration of the PNIPAM core. Concentrated dispersions of these micelles (≥7.5 wt%) showed glassy state behavior below a critical temperature (Tc: 28 °C) which is close to the LCST of the polymers. Below this Tc, the increase in the micelle volume resulted in compression of micelles together above a certain concentration and formation of a glass. We quantified and compared micelle packing at different concentrations and temperatures. The storage moduli (G') of the dispersions showed a universal dependence on the effective volume fraction, which increased substantially above a certain effective volume fraction of φ = 1.2. Furthermore, a disordered lattice model describing this behavior fitted the experimental data and revealed a critical volume fraction of φc = 1.31 close to the experimental value of φ = 1.2. The findings reported provide insights for the molecular design of novel thermosensitive PNIPAM nanoparticles with tunable structural and mechanical properties.

Balancing Enzyme Encapsulation Efficiency and Stability in Complex Coacervate Core Micelles.

Encapsulation of charged proteins into complex coacervate core micelles (C3Ms) can be accomplished by mixing them with oppositely charged diblock copolymers. However, these micelles tend to disintegrate at high ionic strength. Previous research showed that the addition of a homopolymer with the same charge sign as the protein improved the stability of protein-containing C3Ms. In this research, we used fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) to study how the addition of the homopolymer affects the encapsulation efficiency and salt stability of the micelles. We studied the encapsulation of laccase spore coat protein A (CotA), a multicopper oxidase, using a strong cationic-neutral diblock copolymer, poly(N-methyl-2-vinyl-pyridinium iodide)-block-poly(ethylene oxide) (PM2VP128-b-PEO477), and a negatively charged homopolymer, poly(4-styrenesulfonate) (PSS215). DLS indeed showed an improved stability of this three-component C3M system against the addition of salt compared to a two-component system. Remarkably, FCS showed that the release of CotA from a three-component C3M system occurred at a lower salt concentration and over a narrower concentration range than the dissociation of C3Ms. In conclusion, although the addition of the homopolymer to the system leads to micelles with a higher salt stability, CotA is excluded from the C3Ms already at lower ionic strengths because the homopolymer acts as a competitor of the enzyme for encapsulation.

Multicore Liquid Perfluorocarbon-Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking.

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell-labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative 19F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core-shell perfluorocarbon-based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small-angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with 19F MRI and fluorescence imaging, demonstrating their potential for long-term in vivo multimodal imaging.

Native Chemical Ligation for Cross-Linking of Flower-Like Micelles.

In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM- co-HPMA-Cys)-PEG-P(NIPAM- co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM- co-HPMA-ETSA)-PEG-P(NIPAM- co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number ( Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.

Cyclodextrin-based complex coacervate core micelles with tuneable supramolecular host-guest, metal-to-ligand and charge interactions.

Micelles have been recognized as versatile platforms for different biomedical applications, from bioimaging to drug delivery. Complex coacervate core micelles present great advantages compared to traditional micelles, however controlling the number of charges per core-unit and the stability is still a challenge. We here present cyclodextrin-based complex coacervate core micelles where the charge per core-unit can be straightforwardly tuned by cyclodextrin host-guest interactions. By varying the ratio between two adamantane guest molecules, 1-adamantanecarboxylic acid and 1,3-adamantanediacetic acid, the charge of the monomeric core-units can be finely tuned from 6- to 9-. By adding an adamantane bislinker, monomeric core-units can be combined together in dimeric and polymeric structures, increasing the micelles' stability. The orthogonal supramolecular host-guest and coordination-chemistry allows for well-controlled cyclodextrin-based complex coacervate core micelles that offer a versatile platform for designing future, e.g., responsive systems.

Highly cooperative stress relaxation in two-dimensional soft colloidal crystals.

Stress relaxation in crystalline solids is mediated by the formation and diffusion of defects. Although it is well established how externally generated stresses relax, through the proliferation and motion of dislocations in the lattice, it remains relatively unknown how crystals cope with internal stresses. We investigate, both experimentally and in simulations, how highly localized stresses relax in 2D soft colloidal crystals. When a single particle is actively excited, by means of optical tweezing, a rich variety of highly collective stress relaxation mechanisms results. These relaxation processes manifest in the form of open strings of cooperatively moving particles through the motion of dissociated vacancy-interstitial pairs, and closed loops of mobile particles, which either result from cooperative rotations in transiently generated circular grain boundaries or through the closure of an open string by annihilation of a vacancy-interstitial pair. Surprisingly, we find that the same collective events occur in crystals that are excited by thermal fluctuations alone; a large thermal agitation inside the crystal lattice can trigger the irreversible displacements of hundreds of particles. Our results illustrate how local stresses can induce large-scale cooperative dynamics in 2D soft colloidal crystals and shed light on the stabilization mechanisms in ultrasoft crystals.

Nanoparticle-Templated Formation and Growth Mechanism of Curved Protein Polymer Fibrils.

We investigated the growth of biosynthetic protein polymers with templated curvature on pluronic nanospheres. The protein has a central silk-like block containing glutamic residues (S(E)) and collagen-like end-blocks (C). The S(E) blocks stack into filaments when their charge is removed (pH <5). Indeed, at low pH curved and circular fibers are formed at the surface of the nanospheres, which keep their shape after removal of the pluronics. The data reveal the mechanism of the templated fibril-growth: The growth of protein assemblies is nucleated in solution; small protein fibrils adsorb on the nanospheres, presumably due to hydrogen bond formation between the silk-like blocks and the pluronic PEO blocks. The surface of the pluronic particles templates further growth. At relatively low protein/pluronic weight ratios, only a fraction of the nanospheres bears protein fibers, pointing to a limiting amount of nuclei in solution. Because the nanospheres capture fibrils at an early stage of growth, they can be used to separate growth and nucleation rates in protein fibril formation. Moreover, the nanoparticle-templated growth of stable curved fibers opens ways to build proteinaceous nanocapsules from designed protein polymers.

Encapsulation of GFP in Complex Coacervate Core Micelles.

Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms.

Controlling the number of dendrimers in dendrimicelle nanoconjugates from 1 to more than 100.

Herein, we present a facile strategy to controllably build up dendrimicelles by self-assembly of anionic PAMAM dendrimers with cationic-neutral diblock copolymers. We present a systematic study incorporating a full decade (0-9) of dendrimer generations, tracing the gradual variation from aggregates (G0 and G1) to self-assembled micelles (G2-G8), and an unidendrimer micelle structure (G9) by different scattering techniques (light and X-ray). The formed micelles (G2-G9) are spherical in shape with a hydrodynamic radius of about 25 nm. Interestingly, the micellar size, structure and number of incorporated block copolymers are independent of the dendrimer generation (for G2 to G9), while the aggregation number of the dendrimers decreases from 108 to 1, and the stability of the micelles increases upon an increase in the dendrimer generation. Moreover, the micelles with lower generation dendrimers transform from spherical into worm-like structures upon an increase in the positive charge fraction (excess polymers) or ionic strength, while micelles with higher generation dendrimers do not show such a transition. This differential behavior is in-line with a change from a flexible configuration into rigid globular nanoparticles with increasing dendrimer generation. The reported systematic investigation of dendrimicelles comprising a full decade of dendrimer generations provides the basis for versatile strategies focused on building up new (multi)functional materials, e.g. by packing multiple types of dendrimers with different functional groups or encapsulated cargos controllably within one micelle.

Supramolecular assembly of self-healing nanocomposite hydrogels.

Hierarchical self-assembly of transient composite hydrogels is demonstrated through a two-step, orthogonal strategy using nanoparticle tectons interconnected through metal-ligand coordination complexes. The resulting materials are highly tunable with moduli and viscosities spanning many orders of magnitude, and show promising self-healing properties, while maintaining complete optical transparency.

Targeted delivery of TLR ligands to human and mouse dendritic cells strongly enhances adjuvanticity.

Effective vaccines consist of 2 components: immunodominant antigens and effective adjuvants. Whereas it has been demonstrated that targeted delivery of antigens to dendritic cells (DCs) improves vaccine efficacy, we report here that co-targeting of TLR ligands (TLRLs) to DCs strongly enhances adjuvanticity and immunity. We encapsulated ligands for intracellular TLRs within biodegradable nanoparticles coated with Abs recognizing DC-specific receptors. Targeted delivery of TLRLs to human DCs enhanced the maturation and production of immune stimulatory cytokines and the Ag-specific activation of naive CD8(+) T cells. In vivo studies demonstrated that nanoparticles carrying Ag induced cytotoxic T-lymphocyte responses at 100-fold lower adjuvant dose when TLRLs were co-encapsulated instead of administered in soluble form. Moreover, the efficacy of these targeted TLRLs reduced the serum cytokine storm and related toxicity that is associated with administration of soluble TLRLs. We conclude that the targeted delivery of adjuvants may improve the efficacy and safety of DC-based vaccines.

Facile one-step synthesis of monodisperse micron-sized latex particles with highly carboxylated surfaces.

A facile method for the aqueous synthesis of monodisperse and micronmeter-sized colloids with highly carboxylated surfaces is presented. The method is applied to three different monomers, styrene, methyl methacrylate, and 2,2,2-trifluoroethyl methacrylate, and illustrate tuning of the size and monodispersity in the reactions. High surface density of carboxylic acids of up to 10 COOH nm(-2) from potentiometric titrations, is achieved through copolymerization with itaconic acid. The versatility of this system is highlighted by creating highly fluorescent and monodisperse particles that can be index matched in aqueous solution and through surface modification via the carboxylic acid groups using standard amidation chemistry.

Preparation and characterization of low dispersity anionic multiresponsive core-shell polymer nanoparticles.

We prepared anionic multistimuli responsive core-shell polymer nanoparticles with very low size dispersity. By using either acrylic acid (AA) or methacrylic acid (MA) as a comonomer in the poly(N-isopropyl acrylamide) (PNIPAM) shell, we are able to change the distribution of negative charges in the nanoparticle shell. The particle size, volume phase transition temperature, and aggregation state can be modulated using temperature, pH, or ionic strength, providing a very versatile platform for applications in sensors, medical diagnostics, environmental remediation, etc. The nanoparticles have a glassy poly(methyl methacrylate) (PMMA) core of ca. 40 nm radius and a cross-linked PNIPAM anionic shell with either AA or MA comonomers. The particles, p(N-AA) and p(MA-N), respectively, have the same total charge but different charge distributions. While the p(MA-N) particles have the negative charges preferentially distributed toward the inner shell, in the case of the p(N-AA) particles the charge extends more to the particle outer shell. The volume phase transition temperature (T(VPT)) of the particles is affected by the charge distribution and can be fine-tuned by controlling the electrostatic repulsion on the particle shell (using pH and ionic strength). By suppressing the particle charge we can also induce temperature-driven particle aggregation.

On the stability and morphology of complex coacervate core micelles: from spherical to wormlike micelles.

We present a systematic study of the stability and morphology of complex coacervate core micelles (C3Ms) formed from poly(acrylic acid) (PAA) and poly(N-methyl-2-vinylpyridinium)-b-poly(ethylene oxide) (PM2VP-b-PEO). We use polarized and depolarized dynamic and static light scattering, combined with small-angle X-ray scattering, to investigate how the polymer chain length and salt concentration affect the stability, size, and shape of these micelles. We show that C3Ms are formed in aqueous solution below a critical salt concentration, which increases considerably with increasing PAA and PM2VP length and levels off for long chains. This trend is in good agreement with a mean-field model of polyelectrolyte complexation based on the Voorn-Overbeek theory. In addition, we find that salt induces morphological changes in C3Ms when the PAA homopolymer is sufficiently short: from spherical micelles with a diameter of several tens of nanometers at low salt concentration to wormlike micelles with a contour length of several hundreds of nanometers just before the critical salt concentration. By contrast, C3Ms of long PAA homopolymers remain spherical upon addition of salt and shrink slightly. A critical review of existing literature on other C3Ms reveals that the transition from spherical to wormlike micelles is probably a general phenomenon, which can be rationalized in terms of a classical packing parameter for amphiphiles.

Looped structure of flowerlike micelles revealed by 1H NMR relaxometry and light scattering.

We present experimental proof that so-called "flowerlike micelles" exist and that they have some distinctly different properties compared to their "starlike" counterparts. Amphiphilic AB diblock and BAB triblock copolymers consisting of poly(ethylene glycol) (PEG) as hydrophilic A block and thermosensitive poly(N-isopropylacrylamide) (pNIPAm) B block(s) were synthesized via atom transfer radical polymerization (ATRP). In aqueous solutions, both block copolymer types form micelles above the cloud point of pNIPAm. Static and dynamic light scattering measurements in combination with NMR relaxation experiments proved the existence of flowerlike micelles based on pNIPAm(16kDa)-PEG(4kDa)-pNIPAm(16kDa) which had a smaller radius and lower mass and aggregation number than starlike micelles based on mPEG(2kDa)-pNIPAm(16kDa). Furthermore, the PEG surface density was much lower for the flowerlike micelles, which we attribute to the looped configuration of the hydrophilic PEG block. (1)H NMR relaxation measurements showed biphasic T(2) relaxation for PEG, indicating rigid PEG segments close to the micelle core and more flexible distal segments. Even the flexible distal segments were shown to have a lower mobility in the flowerlike micelles compared to the starlike micelles, indicating strain due to loop formation. Taken together, it is demonstrated that self-assemblies of BAB triblock copolymers have their hydrophilic block in a looped conformation and thus indeed adopt a flowerlike conformation.

Quantification of energy input required for chitin nanocrystal aggregate size reduction through ultrasound.

Nanoparticles have been claimed to contribute efficiently to e.g. the mechanical strength of composite materials when present as individual particles. However, these particles tend to aggregate. In this paper we prepare nanocrystals from chitin, a product with high potential added value for application in bio-based materials, and investigate the effect of ultrasound on de-aggregation. Chitin nanocrystals with a length ~ 200 nm and a diameter ~ 15 nm, were obtained via acid hydrolysis of crude chitin powder. Freeze drying resulted in severe aggregation and after redispersion sizes up to ~ 200 µm were found. Ultrasound treatment was applied and break up behaviour was investigated using static light scattering, dynamic light scattering, and laser diffraction. Our results suggest that the cumulative energy input was the dominant factor for chitin nanocrystal aggregate breakup. When a critical energy barrier of ~ 100 kJ/g chitin nanocrystals was exceeded, the chitin nanocrystal aggregates broke down to nanometre range. The break up was mostly a result of fragmentation: the aggregation energy of chitin nanocrystal aggregates was quantified to be ~ 370 kJ/g chitin nanocrystals and we hypothesize that mainly van der Waals interactions and hydrogen bonds are responsible for aggregation.

Deposition behavior of lignin on solid surfaces assessed by stagnation point adsorption reflectometry.

The deposition behavior of lignin on a solid surface was studied using stagnation point adsorption reflectometry (SPAR) comprehensively. In this study, the light absorption coefficient of lignin (τ = 20 476 m-1) at 633 nm wavelength was considered to define a new imaginary refractive index (k) used in the SPAR technique for adsorption analysis. The inclusion of k in the adsorption model yielded an adsorbed amount (Γ) of 1.11 mg m-2, leading to the quality factor (Q f) of 31.71 mg m-2. At a lower concentration, the deposition of lignin on the surface was increased, and it generated a maximum sticking coefficient of ß = 0.71 at 25 mg L-1 on the surface. At the concentration range of 35 and 45 mg L-1, lignin formed clusters and its deposition dropped. The use of Quartz crystal microbalance (QCM) and SPAR techniques also confirmed that the proportion of water in the deposited lignin adlayer was greater when a lower concentration (5-25 mg L-1) of lignin solution was used for adsorption. The findings of this study revealed the potential use of the SPAR technique for evaluating the deposition performance of lignin-based materials on varied surfaces, which would facilitate the development of coating and composite applications for lignin.

Bioflocculants from wastewater: Insights into adsorption affinity, flocculation mechanisms and mixed particle flocculation based on biopolymer size-fractionation.

HYPOTHESIS: Microbial extracellular polymeric substances (EPS) produced from wastewater are generally heterodispersed, which is expected to influence their flocculation performances and mechanism, particularly in mixed particle systems. The different molecular weight (MW) fractions should contribute to the overall adsorption affinity and flocculation mechanism of EPS in single and dual clay systems. EXPERIMENTS: EPS harvested from bioreactors were size-fractionated into high, medium and low MW fractions (HMW, MMW, LMW, respectively). The harvested mixed EPS and its fractions were characterised by diverse analytical techniques coupled with optical reflectometry to investigate the role of each EPS fraction in the overall flocculation mechanism of EPS in kaolinite and montmorillonite clay systems. FINDINGS: In single clay systems, both the harvested mixed EPS and the HMW-EPS fraction showed comparable flocculation performances. However, mixed EPS proved to be more efficient than the HMW-EPS fraction for dual clay flocculation. Site blocking effects were observed in mixed EPS: the LMW and MMW EPS first adsorbed to the surface due to higher diffusivities and faster mass transfer to the interface, while the HMW-EPS were slowly transported but were attached to the surface irreversibly and stronger than the LMW/MMW-EPS. We propose from this, a mixed EPS adsorption mechanism: extended anionic polymer tails in solution, thereby enhancing particle flocculation.

Propagation and attenuation of mechanical signals in ultrasoft 2D solids.

The propagation of elastic waves in soft materials plays a crucial role in the spatiotemporal transmission of mechanical signals, e.g., in biological mechanotransduction or in the failure of marginal solids. At high Reynolds numbers Re ≫ 1, inertia dominates and wave propagation is readily observed. However, mechanical cues in soft and biological materials often occur at low Re, where waves are overdamped. Overdamped waves are not only difficult to observe experimentally, also theoretically their description remains incomplete. Here, we present direct measurements of the propagation and attenuation of mechanical signals in colloidal soft solids, induced by an optical trap. We derive an analytical theory for low Re wave propagation and damping, which is in excellent agreement with the experiments. Our results present both a previously unexplored method to characterize damped waves in soft solids and a theoretical framework showing how localized mechanical signals can provoke a remote and delayed response.