Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer-N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines.

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated N-acylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2-oxazoline)-NH2 (PEtOx-NH2) and characterized by 1H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer-NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer-NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future.

Copper-Binding Properties of Polyethylenimine-Silica Nanocomposite Particles.

Increasing demand for copper resources, accompanied by increasing pollution, has resulted in an urgent need for effective materials for copper binding and extraction. Polyethylenimine (PEI) is one of the strongest copper-chelating agents but is not suitable directly (as is) for most applications due to its high solubility in water. PEI-based composite materials show potential as efficient and practical alternatives. In the present work, the interaction of copper ions with PEI-silica nanocomposite particles and precursor PEI microgels (as a reference) is investigated. It is hypothesized that the main driving force of the reaction is chelation of copper ions by amino groups in the PEI network. The presence of silica in the PEI-silica composites was shown to increase the copper-binding capacity in comparison with the parent microgel. The copper-binding behavior of etched (PEI-free "ghost") composite particles in comparison with the original composites and microgel particles shows that silica nanoparticles in the composite structure increase the number of copper-binding sites in the PEI network rather than adsorbing copper themselves. PEI-silica composites can be easily recycled after copper adsorption by simply washing in 1 M nitric acid, which results in complete copper extraction. Employing this recovery method, PEI-silica composite particles can be used for multiple, efficient cycles of copper removal and extraction.

Tuning Cellular Interactions of Carboxylic Acid-Side-Chain-Containing Polyacrylates: The Role of Cyanine Dye Label and Side-Chain Type.

Cellular uptake and intracellular targeting to specific organelles are key events in the cellular processing of nanomaterials. Herein, we perform a detailed structure-property relationship study on carboxylic acid-side-chain-bearing polyacrylates to provide design criteria for the manipulation of their cellular interactions. Redox-initiated reversible addition-fragmentation chain-transfer (RRAFT) polymerization of three tert-butyl-protected N-acylated amino ester-based acrylate monomers of different substitutions and degrees of polymerization (DPs) yielded defined and pH-responsive carboxylic acid-side-chain polymers upon deprotection (N-acetyl, DP 1: P(M1); N-propionyl, DP 1: P(E1), DP 2: P(E2)). Flow cytometry studies revealed time-dependent cell association with P(E2) > P(E1) > P(M1) at any given time point. Importantly, the type of cyanine dye used for labeling was found to significantly influence the cellular processing of the polymers. Changing the dye from Cy5 to its sulfonated version sulfoCy5 resulted in a much lower cellular association. Moreover, Cy5-labeled polymers were targeted to mitochondria, while sulfoCy5 modification caused a significant change in the cellular fate of polymers toward lysosome trafficking. This study highlights the importance of selecting a suitable dye but also demonstrates the possibilities for the rational design of organelle-specific targeting of carboxylated polyacrylates.

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding.

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.

Attachment of Salmonella strains to a plant cell wall model is modulated by surface characteristics and not by specific carbohydrate interactions.

BACKGROUND: Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface. RESULTS: We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin. CONCLUSIONS: Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils.

Cyclic RGDfK Peptide Functionalized Polymeric Nanocarriers for Targeting Gemcitabine to Ovarian Cancer Cells.

Current cancer chemotherapies commonly suffer from nonspecificity, drug resistance, poor bioavailability, and narrow therapeutic indices. To achieve the optimum drug efficacy, we designed a polymeric drug delivery system for targeted intracellular delivery of a clinically approved, water-soluble anticancer drug, gemcitabine hydrochloride (GEM). We utilized the unique ability of a cyclic pentapeptide cRGDfK to specifically target αvß3 integrin receptors that are overexpressed on SKOV-3 human ovarian cancer cells. This significantly increased the effective intracellular drug concentration even at low doses, thereby remarkably improving the chemotherapeutic potential of GEM. cRGDfK-conjugated, GEM-loaded nanoparticles reduced the nonspecific hemolytic cytotoxicity of the drug, simultaneously influencing intracellular processes such as mitochondrial membrane potential (DΨm), reactive oxygen species (ROS) levels, and apoptosis, thereby favorably influencing drug antiproliferative efficacy.

Mesoporous, anisotropic nanostructures from bioinspired polymeric catecholamine neurotransmitters and their potential application as photoacoustic imaging agents.

Mesoporous polydopamine (PDA) nanobowls, which can be prepared using Pluronic® F-127, ammonia, and 1,3,5-trimethylbenzene (TMB), are one of the most studied anisotropic nanoparticle systems. However, only limited reports on polymerised analogues polynorepinephrine (PNE) and polyepinephrine (PEP) exist. Herein, we present modifications to a one-pot, soft template method, originally applied to make PDA nanobowls, to fabricate new shape-anisotropic nanoparticles (mesoporous nanospheres or "nano-golf balls" and nanobowls) using PNE and PEP for the first time. These modifications include the use of different oil phases (TMB, toluene and o-xylene) and ammonia concentrations to induce anisotropic growth of PDA, PNE, and PEP particles. Moreover, this work features the application of oddly shaped PDA, PNE, and PEP nanoparticles as intravascular photoacoustic imaging enhancers in Intralipid®-India ink-based tissue-mimicking phantoms. Photoacoustic imaging experiments showed that mesoporous nanobowls exhibit stronger enhancement, in comparison to their mesoporous nano-golf ball and nanoaggregate counterparts. The photoacoustic enhancement also followed the general trend PDA > PNE > PEP due to the differences in the rates of polymerisation of the monomers and the optical absorption of the resulting polymers. Lastly, about two- to four-fold enhancement in photoacoustic signals was observed for the mesoporous nanostructures, when compared to smooth nanospheres and their nano-aggregates. These results suggest that shape manipulation can aid in overcoming the inherently lower performance of PNE and PEP as photoacoustic imaging agents, compared to PDA. Since nanomaterials with mesoporous and anisotropic morphologies have significant, unexplored potential with emerging applications, these results set the groundwork for future studies on photoacoustically active oddly shaped PNE- and PEP-based nanosystems.

Enhanced photoacoustic imaging in tissue-mimicking phantoms using polydopamine-shelled perfluorocarbon emulsion droplets.

The current work features process parameters for the ultrasound (25 kHz)-assisted fabrication of polydopamine-shelled perfluorocarbon (PDA/PFC) emulsion droplets with bimodal (modes at 100-600 nm and 1-6 µm) and unimodal (200-600 nm) size distributions. Initial screening of these materials revealed that only PDA/PFC emulsion droplets with bimodal distributions showed photoacoustic signal enhancement due to large size of their optically absorbing PDA shells. Performance of this particular type of emulsion droplets as photoacoustic agents were evaluated in Intralipid®-India ink media, mimicking the optical scattering and absorbanceof various tissuetypes. From these measurements, it was observed that PDA/PFC droplets with bimodal size distributions can enhance the photoacoustic signal of blood-mimicking phantom by up to five folds in various tissue-mimicking phantoms with absorption coefficients from 0.1 to 1.0 cm-1. Furthermore, using the information from enhanced photoacoustic images at 750 nm, the ultimate imaging depth was explored for polydopamine-shelled, perfluorohexane (PDA/PFH) emulsion droplets by photon trajectory simulations in 3D using a Monte Carlo approach. Based on these simulations, maximal tissue imaging depths for PDA/PFH emulsion droplets range from 10 to 40 mm, depending on the tissue type. These results demonstrate for the first time that ultrasonically fabricated PDA/PFC emulsion droplets have great potential as photoacoustic imaging agents that can be complemented with other reported characteristics of PDA/PFC emulsion droplets for extended applications in theranostics and other imaging modalities.

Tracking the heat-triggered phase change of polydopamine-shelled, perfluorocarbon emulsion droplets into microbubbles using neutron scattering.

Perfluorocarbon emulsion droplets are hybrid colloidal materials with vast applications, ranging from imaging to drug delivery, due to their controllable phase transition into microbubbles via heat application or acoustic droplet vapourisation. The current work highlights the application of small- and ultra-small-angle neutron scattering (SANS and USANS), in combination with contrast variation techniques, in observing the in situ phase transition of polydopamine-shelled, perfluorocarbon (PDA/PFC) emulsion droplets with controlled polydispersity into microbubbles upon heating. We correlate these measurements with optical and transmission electron microscopy imaging, dynamic light scattering, and thermogravimetric analysis to characterise these emulsions, and observe their phase transition into microbubbles. Results show that the phase transition of PDA/PFC droplets with perfluorohexane (PFH), perfluoropentane (PFP), and PFH-PFP mixtures occur at temperatures that are around 30-40 °C higher than the boiling points of pure liquid PFCs, and this is influenced by the specific PFC compositions (perfluorohexane, perfluoropentane, and mixtures of these PFCs). Analysis and model fitting of neutron scattering data allowed us to monitor droplet size distributions at different temperatures, giving valuable insights into the transformation of these polydisperse, emulsion droplet systems.

Synthesis and characterisation of polynorepinephrine-shelled microcapsules via an oil-in-water emulsion templating route.

In this article, we present a facile and robust method for the surfactant-free preparation of polynorepinephrine stabilised microcapsules templated from an oil-in-water emulsion. The resulting microcapsule structures are dependent on the concentration of Cu2+ used to catalyse norepinephrine polymerisation. When the concentration of Cu2+ increases, the diameter of the microcapsules and the thickness of the shell increase correspondingly. The mechanical and chemical stability provided by the polynorepinephrine shell are explored using surface pressure measurements and atomic force microscopy, demonstrating that a rigid and robust polynorepinephrine shell is formed. In order to demonstrate potential application of the microcapsules in sustained release, Nile red stained squalane was encapsulated, and pH responsive release was monitored. It was seen that by controlling pH, the release profile could be controlled, with highest release efficacy achieved in alkaline conditions, offering a new pathway for development of encapsulation systems for the delivery of water insoluble actives.

Photothermally responsive Pickering emulsions stabilised by polydopamine nanobowls.

Pickering emulsions with stimuli responsive properties have attracted mounting research attention owing to their potential for on-demand destabilisation of emulsions. However, a combination of biocompatibility and long-term stability are essential to efficiently apply such systems in biomedical applications, and this remains a significant challenge. To address current limitations, here we report the formation of photothermally responsive oil-in-water (o/w) Pickering emulsions fabricated using biocompatible stabilisers and showing prolonged stability. For the first time, we explore polydopamine (PDA) bowl-shaped mesoporous nanoparticles (PDA nanobowls) as a Pickering stabiliser without any surface modification or other stabiliser present. As-prepared PDA nanobowl-stabilised Pickering emulsions are shown to be pH responsive, and more significantly show high photothermal efficiency under near-infrared illumination due the incorporation of PDA into the system, which has remarkable photothermal response. These biocompatible, photothermally responsive o/w Pickering emulsion systems show potential in controlled drug release applications stimulated by NIR illumination.

Open-Closed Structure of Light-Responsive Protein LOV2 Regulates Its Molecular Interaction with a Binding Partner.

Optogenetic approaches have broad applications, including regulating cell signaling and gene expression. Photoresponsive protein LOV2 and its binding partner ZDK represent an important protein caging/uncaging optogenetic system. Herein, we combine time-resolved small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) to reveal different structural states of LOV2 and the light-controlled mechanism of interaction between LOV2 and ZDK. In response to blue light within a time frame of ca. 70 s, LOV2 has a significantly higher value of radius of gyration Rg (29.6 ± 0.3 vs 26.4 ± 0.4 Å) than its dark state, suggesting unwinding of the C-terminal Jα-helix into an open structure. Atomic force microscopy was used to characterize molecular interactions of LOV2 in open and closed states with ZDK at a single-molecule level. The closed state of LOV2 enables strong binding with ZDK, characterized by a 60-fold lower dissociation rate and a ∼1.5-times higher activation energy barrier than for its open state. In combination, these data support a light-switching mechanism that is modulated by the proximity of multiple binding sites of LOV2 for ZDK.

Bioinspired polynorepinephrine nanoparticles as an efficient vehicle for enhanced drug delivery.

An innovative drug delivery vehicle based on polynorepinephrine (PNE) with controllable size modification, high delivery efficacy and low cytotoxicity is presented. Highly monodisperse PNE nanoparticles are fabricated by the autoxidation of norepinephrine monomers in an alkaline water/ethanol mixture via stirring at room temperature. We demonstrated the facile optimization of particle size to enhance particle stability and biocompatibility by varying solvent and monomer dosage. To demonstrate the suitability and potential application of PNE particles in cancer therapy, we show that these particles are biocompatible in vitro with HeLa cells and in vivo in zebrafish embryos. After loading the anti-cancer chemotherapy drug doxorubicin (DOX) into the PNE nanoparticles, a consistent and pH responsive drug release profile of DOX was achieved in different environmental conditions. It was found that DOX loaded PNE nanoparticles (PNE/DOX) exhibit much higher pharmaceutical cytotoxicity than free DOX on HeLa cells. Furthermore, the amount of drug released was significantly enhanced in acidic environments that mimic the pH of extracellular tumour microenvironments. Taken together, the PNE nanoparticles represent a new class of melanin particles with promising potential in drug delivery and as a therapeutic platform for cancer treatment.

Effect of protein adsorption on the radial wicking of blood droplets in paper.

HYPOTHESES: (1) The equilibrium size and characteristics of a radially wicked fluid on porous material such as paper is expected to be dependent on the fluid properties and therefore could serve as a diagnostic tool. (2) The change in wicked stain size between biological fluids is dependent on a change in solid-liquid surface interfacial energy due to protein adsorption. EXPERIMENTS: Sessile droplets of increasing volume of blood, its components, and model fluids were deposited onto paper and the equilibrium stain size after coming to a halt was recorded. The contact angle of fluid droplets on model cellulose surfaces was measured to quantify the effect that blood protein adsorption at the solid-liquid interface has on radially wicked equilibrium size. Finally the significance of droplet evaporation for the time scale of interest was analysed. FINDINGS: The final stain area of all fluids tested on paper scales remarkably linearly with droplet volume. Different fluids were compared and the gradient of this linear relation was measured. Model fluids varying in surface tension and viscosity all behave similarly and exhibit a constant gradient. Blood and its components produce smaller stains, demonstrated by lower gradients. The gradient is a function of protein concentration, thus the mechanism of this phenomenon was identified as protein adsorption at the cellulose-liquid interface. The slope of the area/volume relationship for droplets is an important quantitative mechanistic variable.

Lubricin: a versatile, biological anti-adhesive with properties comparable to polyethylene glycol.

Lubricin is a glycoprotein found in articular joints which has been recognized as being an important biological boundary lubricant molecule. Besides providing lubrication, we demonstrate, using a quartz crystal microbalance, that lubricin also exhibits anti-adhesive properties and is highly effective at preventing the non-specific adsorption of representative globular proteins and constituents of blood plasma. This impressive anti-adhesive property, combined with lubricin's ability to readily self-assemble to form dense, highly stable telechelic polymer brush layers on virtually any substrates, and its innate biocompatibility, makes it an attractive candidate for anti-adhesive and anti-fouling coatings. We show that coatings of lubricin protein are as effective as, or better than, self-assembled monolayers of polyethylene glycol over a wide range of pH and that this provides a simple, versatile, highly stable, and highly effective method of controlling unwanted adhesion to surfaces.

Adsorption of cationic polyacrylamide at the cellulose-liquid interface: a neutron reflectometry study.

The layer thickness and density of high molecular weight cationic polyacrylamide (CPAM) adsorbed at the cellulose-water interface was quantified by neutron reflectometry. The thickness of a full monolayer of CPAM of constant molecular weight (13 MD) but different charge densities, adsorbed with or without NaCl (10(-3) M), was studied. Thin cellulose films (40±7 Å) of roughness <10 Å were produced by spin coating a cellulose acetate-acetone solution and regenerating by alkaline hydrolysis. Film smoothness was greatly improved by controlling the concentration of cellulose acetate (0.13 wt%) and the hydrolysis time in sodium methoxide. The adsorption thickness of CPAM (40% charge 13 MD) at the solid-D2O interface was 43±4 Å on cellulose and 13±2 Å on silicon, an order of magnitude smaller than the CPAM radius of gyration. At constant molecular weight, the thickness of the CPAM layer adsorbed on cellulose increases with polymer charge density (10±1 Å at 5%). Addition of 10(-3) M NaCl decreased the thickness of CPAM layer already adsorbed on cellulose. However, the adsorption layer on cellulose of a CPAM solution equilibrated in 10(-3) M NaCl is much thicker (89±11 Å for 40% CPAM). For high molecular weight CPAMs adsorbed from solution under constant conditions, the adsorption layer can be varied by 1 order of magnitude via control of the variables affecting electrostatic intra- and inter-polymer chain interactions.