An Inverse Thermogelling Bioink Based on an ABA-Type Poly(2-oxazoline) Amphiphile.

Hydrogels are key components in several biomedical research areas such as drug delivery, tissue engineering, and biofabrication. Here, a novel ABA-type triblock copolymer comprising poly(2-methyl-2-oxazoline) as the hydrophilic A blocks and poly(2-phenethyl-2-oxazoline) as the aromatic and hydrophobic B block is introduced. Above the critical micelle concentration, the polymer self-assembles into small spherical polymer micelles with a hydrodynamic radius of approx 8-8.5 nm. Interestingly, this specific combination of hydrophilic and hydrophobic aromatic moieties leads to rapid thermoresponsive inverse gelation at polymer concentrations above a critical gelation concentration (20 wt %) into a macroporous hydrogel of densely packed micelles. This hydrogel exhibited pronounced viscoelastic solid-like properties, as well as extensive shear-thinning, rapid structure recovery, and good strain resistance properties. Excellent 3D-printability of the hydrogel at lower temperature opens a wide range of different applications, for example, in the field of biofabrication. In preliminary bioprinting experiments using NIH 3T3 cells, excellent cell viabilities of more than 95% were achieved. The particularly interesting feature of this novel material is that it can be used as a printing support in hybrid bioink systems and sacrificial bioink due to rapid dissolution at physiological conditions.

Monocytes Represent One Source of Bacterial Shielding from Antibiotics following Influenza Virus Infection.

Methicillin-resistant Staphylococcus aureus has emerged as a significant contributor to morbidity and mortality associated with influenza infection. In this study, we show in a mouse model that preceding influenza infection promotes S. aureus resistance to killing by antibiotics. This resistance coincides with influenza-induced accumulation of inflammatory monocytes in the lung. CCR type 2 (CCR2) is responsible for pulmonary monocyte recruitment after influenza infection. We found that antibiotic-treated Ccr2-deficient (Ccr2-/-) mice exhibit significantly improved bacterial control and survival from influenza and methicillin-resistant S. aureus coinfection, despite a delay in viral clearance. Mechanistically, our results from in vivo studies indicate that influenza-induced monocytes serve as reservoirs for intracellular S. aureus survival, thereby promoting bacterial resistance to antibiotic treatment. Blocking CCR2 with a small molecular inhibitor (PF-04178903), in conjunction with antibiotic treatment, enhanced lung bacterial clearance and significantly improved animal survival. Collectively, our study demonstrates that inflammatory monocytes constitute an important and hitherto underappreciated mechanism of the conflicting immune requirements for viral and bacterial clearance by hosts, which subsequently leads to exacerbated outcomes of influenza and S. aureus coinfection.

A model for in situ plan of care for a critically unstable pediatric patient following I-131 MIBG infusion.

Recent clinical trials have moved iodine-131 (I-131) metaiodobenzylguanidine (MIBG) therapy into frontline management of high-risk neuroblastoma. With this expansion, it is reasonable to anticipate the need for intensive care level resuscitations. Radiation exposure remains the greatest risk to health care professionals managing these patients. We combined shock simulation scenario data with actual radiation dosimetry data to create a care model allowing for aggressive, prolonged in situ resuscitation of a critically ill pediatric patient after I-131 MIBG administration. This model will maintain a critical care provider's radiation level below 10% of the annual occupational dose limit (5 mSv, 500 mrem) per patient managed.

Origin of the low-frequency plateau and the light-scattering slow mode in semidilute poly(ethylene glycol) solutions.

A low-frequency plateau is often found in the rheological spectra of various kinds of semidilute solutions of polymers and other colloids; also, many such solutions have been reported to show slow-modes in their dynamic light scattering autocorrelation functions. Both these observations may lead to the hypothesis of weak associative network structures built by the dissolved polymer chains or colloidal building blocks. To challenge this hypothesis, we conduct a series of comparative studies on semidilute solutions of poly(ethylene glycol) by using classical rheology as well as passive microrheology based on dynamic light scattering, along with structural studies using static light scattering. Although we indeed find a low-frequency plateau using classical shear rheology, even at elevated temperatures where potential polymer aggregates should be broken, no such plateau is observed in any of our microrheology experiments. Also, dynamic and static light scattering studies on the polymer solutions do not confirm the presence of larger structural entities: no slow mode can be detected in the autocorrelation function of the scattering intensity signal, and this signal is angle independent if the samples are purified by a thorough procedure of filtration. Based on these findings, we conclude that the low-frequency plateau in classical rheology results is an instrument effect caused by erroneous recording of the phase angle, although the magnitude of the torque lies well within the resolution of the rheometer. We also conclude that slow modes in dynamic light scattering on solutions of poly(ethylene glycol) are impurity-based artifacts rather than due to actual associated structures.

Protein corona-mediated targeting of nanocarriers to B cells allows redirection of allergic immune responses.

BACKGROUND: Nanoparticle (NP)-based vaccines are attractive immunotherapy tools because of their capability to codeliver antigen and adjuvant to antigen-presenting cells. Their cellular distribution and serum protein interaction ("protein corona") after systemic administration and their effect on the functional properties of NPs is poorly understood. OBJECTIVES: We analyzed the relevance of the protein corona on cell type-selective uptake of dextran-coated NPs and determined the outcome of vaccination with NPs that codeliver antigen and adjuvant in disease models of allergy. METHODS: The role of protein corona constituents for cellular binding/uptake of dextran-coated ferrous nanoparticles (DEX-NPs) was analyzed both in vitro and in vivo. DEX-NPs conjugated with the model antigen ovalbumin (OVA) and immunostimulatory CpG-rich oligodeoxynucleotides were administered to monitor the induction of cellular and humoral immune responses. Therapeutic effects of this DEX-NP vaccine in mouse models of OVA-induced anaphylaxis and allergic asthma were assessed. RESULTS: DEX-NPs triggered lectin-induced complement activation, yielding deposition of activated complement factor 3 on the DEX-NP surface. In the spleen DEX-NPs targeted predominantly B cells through complement receptors 1 and 2. The DEX-NP vaccine elicited much stronger OVA-specific IgG2a production than coadministered soluble OVA plus CpG oligodeoxynucleotides. B-cell binding of the DEX-NP vaccine was critical for IgG2a production. Treatment of OVA-sensitized mice with the DEX-NP vaccine prevented induction of anaphylactic shock and allergic asthma accompanied by IgE inhibition. CONCLUSIONS: Opsonization of lectin-coated NPs by activated complement components results in selective B-cell targeting. The intrinsic B-cell targeting property of lectin-coated NPs can be exploited for treatment of allergic immune responses.

Resistance to Acute Macrophage Killing Promotes Airway Fitness of Prevalent Community-Acquired Staphylococcus aureus Strains.

The incidence of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in otherwise healthy individuals is increasing. To investigate the mechanism underlying the epidemiological success of predominant community-associated (CA)-MRSA strains, we examined their fitness traits during the initial interaction between bacteria and the host occurring in the lower airway. Using a mouse respiratory infection model, we show that clinical isolates often responsible for CA infections are highly resistant to clearance from healthy airways, whereas S. aureus strains not as prevalent or traditionally associated with hospital-associated infections are relatively susceptible. Mechanistically, the competitive fitness of S. aureus is a result of both agr-dependent and -independent resistance to innate bacterial killing. Furthermore, we show that rather than evasion from neutrophil-dependent bactericidal process, the observed S. aureus fitness in the lower airways is due to its intrinsic resistance to resident alveolar macrophage-mediated intracellular killing. Importantly, we demonstrate that the virulence determinants responsible for bacterial persistence in immune-competent mice are dispensable in mice with predisposing conditions such as influenza infection. Taken together, these novel findings of the improved competence of predominant CA-MRSA strains to survive innate killing in healthy hosts, particularly at the very beginning stage of infection, provide a unique insight into their epidemiological success.

Is RuAs2 a candidate for high temperature thermoelectric applications?

The mineral inspired material RuAs2 shows promise as a thermoelectric material with its high stability and attractive band structure. In order to validate these expectations phase-pure polycrystalline ruthenium arsenide was synthesized and densified using Spark Plasma Sintering. RuAs2 is an n-type semiconductor with an indirect band gap 0.69 eV as estimated from temperature dependent resistivity data, while the band gap calculated with DFT is 0.64 eV. The thermal conductivity and electrical resistivity are both high with room temperature values of 16 W m-1 K-1 and 170 mΩ cm respectively, leading to modest thermoelectric properties for the intrinsic system. Band structure calculations suggest that chemical modification should preferably be done at the As site to improve the intrinsic properties. Synchrotron powder X-ray diffraction and Rietveld structural refinements show RuAs2 to be a stable line phase up to 1000 K in both in air and in vacuum, and both as a powder and as a dense pellet. No indication of preferential orientation or material gradients are observed.

Low-Temperature Anharmonicity in Cesium Chloride (CsCl).

Anharmonic lattice vibrations govern heat transfer in materials, and anharmonicity is commonly assumed to be dominant at high temperature. The textbook cubic ionic defect-free crystal CsCl is shown to have an unexplained low thermal conductivity at room temperature (ca. 1 W/(m K)), which increases to around 13  W/(m K) at 25 K. Through high-resolution X-ray diffraction it is unexpectedly shown that the Cs atomic displacement parameter becomes anharmonic at 20 K.

Oxidation-Responsive and "Clickable" Poly(ethylene glycol) via Copolymerization of 2-(Methylthio)ethyl Glycidyl Ether.

Poly(ethylene glycol) (PEG) is a widely used biocompatible polymer. We describe a novel epoxide monomer with methyl-thioether moiety, 2-(methylthio)ethyl glycidyl ether (MTEGE), which enables the synthesis of well-defined thioether-functional poly(ethylene glycol). Random and block mPEG-b-PMTEGE copolymers (Mw/Mn = 1.05-1.17) were obtained via anionic ring opening polymerization (AROP) with molecular weights ranging from 5 600 to 12 000 g·mol(-1). The statistical copolymerization of MTEGE with ethylene oxide results in a random microstructure (rEO = 0.92 ± 0.02 and rMTEG E = 1.06 ± 0.02), which was confirmed by in situ (1)H NMR kinetic studies. The random copolymers are thermoresponsive in aqueous solution, with a wide range of tunable transition temperatures of 88 to 28 °C. In contrast, mPEG-b-PMTEGE block copolymers formed well-defined micelles (Rh ≈ 9-15 nm) in water, studied by detailed light scattering (DLS and SLS). Intriguingly, the thioether moieties of MTEGE can be selectively oxidized into sulfoxide units, leading to full disassembly of the micelles, as confirmed by detection of pure unimers (DLS and SLS). Oxidation-responsive release of encapsulated Nile Red demonstrates the potential of these micelles as redox-responsive nanocarriers. MTT assays showed only minor effects of the thioethers and their oxidized derivatives on the cellular metabolism of WEHI-164 and HEK-293T cell lines (1-1000 µg·mL(-1)). Further, sulfonium PEG polyelectrolytes can be obtained via alkylation or alkoxylation of MTEGE, providing access to a large variety of functional groups at the charged sulfur atom.

Physicochemical and Preclinical Evaluation of Spermine-Derived Surfactant Liposomes for in Vitro and in Vivo siRNA-Delivery to Liver Macrophages.

Herein we report on a liposomal system for siRNA delivery consisting of cholesterol (Chol), distearoylphosphatidylcholine (DSPC), and surfactant TF (1-hydroxy-50-amino-3,4,7,10,13,16,19,22-octaoxa-37,41,45-triaza-pentacontane), a novel spermine derivative (HO-EG8-C12-spermine) which has shown improved siRNA delivery to cells in vitro and in vivo. Predominantly single-walled liposomes with reproducible sizes and moderately broad size distributions were generated with an automated extrusion device. The liposomes remained stable when prepared in the presence of siRNA at N/P ratios of 17-34. However, when mixed with human serum in equal volumes, larger aggregates in the size range of several hundred nanometers were observed by dynamic light scattering. These larger aggregates could potentially limit prolonged in vivo applications. Aggregate formation could be reduced by the addition of a cholesterol-hyperbranched polyglycerol surfactant (hbPG) that sterically shields the liposomal surface against serum induced aggregation. In vitro experiments with murine macrophages utilizing macrophage-specific anti-CD68 siRNA loaded liposomes showed potent and sequence specific reduction of CD68 transcript levels without cytotoxicity. Experiments in mice using intravenous application of CW800 NHS ester labeled liposomes, near-infrared in vivo imaging, and fluorescent assisted cell sorting of inflammatory cells demonstrated an almost quantitative accumulation of these liposomes, with and without hbPG, in the liver and a specific knockdown of CD68 mRNA of up to 70% in liver resident macrophages. It was found that aggregate formation of TF liposomes in serum does not significantly affect in vivo siRNA delivery to these central inflammatory cells of the liver.

Functionalization of Active Ester-Based Polymersomes for Enhanced Cell Uptake and Stimuli-Responsive Cargo Release.

Poly(2,3-dihydroxypropyl methacrylamide) (P(DHPMA))-based amphiphilic block copolymers have recently proven to form polymer vesicles (polymersomes). In this work, we further expand their potential by incorporating (i) units for pH-dependent disintegration into the hydrophobic membrane and (ii) mannose as targeting unit into the hydrophilic block. This last step relies on the use of an active ester prepolymer. We confirm the stability of the polymersomes against detergents like Triton X-100 and their low cytotoxicity. The incorporation of 2-(2,2-dimethyl-1,3-dioxolane-4-yl)ethyl methacrylate into the hydrophobic block (lauryl methacrylate) allows a pH-responsive disintegration for cargo release. Efficient decomposition of the polymersome structure is monitored by dynamic light scattering. It is thus possible to include an active enzyme (glucose oxidase), which gets only active (is set free) after vesicle disintegration. In addition, the introduction of mannose as targeting structure allows enhanced and selective targeting of dendritic cells.

Pentafluorophenyl Ester-based Polymersomes as Nanosized Drug-Delivery Vehicles.

In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption-induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.

Immunomodulatory Function of Interleukin 28B during primary infection with cytomegalovirus.

BACKGROUND: Feedback mechanisms between interferons α and λ (IFNs) may be affected by single nucleotide polymorphisms (SNP) in interleukin 28B (IL-28B; IFN-λ3) promoter region and may influence cytomegalovirus (CMV) replication. METHODS: We associated IL-28B SNPs with the risk of CMV replication after transplantation. Next, we examined the effect of IL-28B genotypes on IL-28B, and IFN-stimulated gene (ISG) expression, and CMV replication in human foreskin fibroblast (HFF) and peripheral blood mononuclear cells (PBMCs). RESULTS: Transplant recipients with an IL-28B SNP (rs8099917) had significantly less CMV replication (P = .036). Both HFF-cells and PBMCs with a SNP showed lower IL-28B expression during infection with CMV, but higher "antiviral" ISG expression (eg, OAS1). Fibroblasts with a SNP had a 3-log reduction of CMV replication at day 4 (P = .004). IL-28B pretreatment induced ISG expression in noninfected fibroblasts, but a relative decrease of ISG expression could be observed in CMV-infected fibroblasts. The inhibitory effects of IL-28B could be abolished by siRNA or antagonistic peptides against the IL-28 receptor. In fibroblasts, inhibition of IL-28 signaling resulted in an increase of ISG expression and 3-log reduction of CMV-replication (P = .01). CONCLUSIONS: We postulate that IL-28B may act as a key regulator of ISG expression during primary CMV infection. IL-28B SNPs may be associated with higher antiviral ISG expression, which results in better replication control.

Multifunctional two-photon active silica-coated Au@MnO Janus particles for selective dual functionalization and imaging.

Monodisperse multifunctional and nontoxic Au@MnO Janus particles with different sizes and morphologies were prepared by a seed-mediated nucleation and growth technique with precise control over domain sizes, surface functionalization, and dye labeling. The metal oxide domain could be coated selectively with a thin silica layer, leaving the metal domain untouched. In particular, size and morphology of the individual (metal and metal oxide) domains could be controlled by adjustment of the synthetic parameters. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g., antibodies, proteins) in a single step for converting the photoluminescent and superparamagnetic Janus nanoparticles into multifunctional efficient vehicles for theranostics. The Au@MnO@SiO2 Janus particles were characterized using high-resolution transmission electron microscopy (HR-)TEM, powder X-ray diffraction (PXRD), optical (UV-vis) spectroscopy, confocal laser fluorescence scanning microscopy (CLSM), and dynamic light scattering (DLS). The functionalized nanoparticles were stable in buffer solution or serum, showing no indication of aggregation. Biocompatibility and potential biomedical applications of the Au@MnO@SiO2 Janus particles were assayed by a cell viability analysis by coincubating the Au@MnO@SiO2 Janus particles with Caki 1 and HeLa cells. Time-resolved fluorescence spectroscopy in combination with CLSM revealed the silica-coated Au@MnO@SiO2 Janus particles to be highly two-photon active; no indication for an electronic interaction between the dye molecules incorporated in the silica shell surrounding the MnO domains and the attached Au domains was found; fluorescence quenching was observed when dye molecules were bound directly to the Au domains.

Selective uptake of cylindrical poly(2-oxazoline) brush-antiDEC205 antibody-OVA antigen conjugates into DEC-positive dendritic cells and subsequent T-cell activation.

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well-defined structures with a high biological specificity. A polymeric nanocarrier is presented that consists of a cylindrical brush polymer with poly-2-oxazoline side chains carrying an azide functional group on each of the many side chain ends. After click conjugation of dye and an anti-DEC205 antibody to the periphery of the cylindrical brush polymer, antibody-mediated specific binding and uptake into DEC205(+) -positive mouse bone marrow-derived dendritic cells (BMDC) was observed, whereas binding and uptake by DEC205(-) negative BMDC and non-DC was essentially absent. Additional conjugation of an antigen peptide yielded a multifunctional polymer structure with a much stronger antigen-specific T-cell stimulatory capacity of pretreated BMDC than application of antigen or polymer-antigen conjugate.

Aggregation behavior of cationic nanohydrogel particles in human blood serum.

For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential for the complex. Preliminary in vivo studies confirmed the results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe and stable complexes at a charge neutralized ratio and thus making them applicable to systemic siRNA delivery.

Hybrid assemblies based on a gadolinium-containing polyoxometalate and a cationic polymer with spermine side chains for enhanced MRI contrast agents.

Supramolecular assembly: Spherical and stable hybrid assemblies based on a cationic polymer with spermine side chains and an anionic Gd(3+)-containing polyoxometalate cluster (GdW) are prepared by electrostatic interaction. The T1-weighted MRI performance of GdW is enhanced about three times in the assemblies; meanwhile, the assemblies show good biocompatibility, which enables them to be promising candidates for MRI contrast agents.

Interaction of pHPMA-pLMA copolymers with human blood serum and its components.

Immediately after administration, polymer therapeutics are exposed to complex biological media like blood which may influence and alter their physicochemical properties due to interactions with proteins or serum components. Among such interactions those leading to larger sized aggregates can be sensitively detected by dynamic light scattering (DLS) as a pre in vivo screening method. Random copolymers from N-(2-hydroxypropyl)methacrylamide and lauryl methacrylate p(HPMA-co-LMA) and copolymers loaded with the model drug domperidone were characterized by DLS in isotonic salt solution and in blood serum. The bare amphiphilic copolymer micelles (Rh=30 nm in isotonic salt solution) formed large aggregates in serum of over 100 nm radius which were shown to originate from interactions with very low density lipoproteins (VLDLs). Encapsulation of the hydrophobic drug domperidone resulted, at first, in drug-copolymer formulations with larger hydrodynamic radii (39 nm

PAA-PAMPS copolymers as an efficient tool to control CaCO3 scale formation.

Scale formation, the deposition of certain minerals such as CaCO3, MgCO3, and CaSO4·2H2O in industrial facilities and household devices, leads to reduced efficiency or severe damage. Therefore, incrustation is a major problem in everyday life. In recent years, double hydrophilic block copolymers (DHBCs) have been the focus of interest in academia with regard to their antiscaling potential. In this work, we synthesized well-defined blocklike PAA-PAMPS copolymers consisting of acrylic acid (AA) and 2-acrylamido-2-methyl-propane sulfonate (AMPS) units in a one-step reaction by RAFT polymerization. The derived copolymers had dispersities of 1.3 and below. The copolymers have then been investigated in detail regarding their impact on the different stages of the crystallization process of CaCO3. Ca(2+) complexation, the first step of a precipitation process, and polyelectrolyte stability in aqueous solution have been investigated by potentiometric measurements, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS). A weak Ca(2+) induced copolymer aggregation without concomitant precipitation was observed. Nucleation, early particle growth, and colloidal stability have been monitored in situ with DLS. The copolymers retard or even completely suppress nucleation, most probably by complexation of solution aggregates. In addition, they stabilize existing CaCO3 particles in the nanometer regime. In situ AFM was used as a tool to verify the coordination of the copolymer to the calcite (104) crystal surface and to estimate its potential as a growth inhibitor in a supersaturated CaCO3 environment. All investigated copolymers instantly stopped further crystal growth. The carboxylate richest copolymer as the most promising antiscaling candidate proved its enormous potential in scale inhibition as well in an industrial-filming test (Fresenius standard method).

Collapse of cylindrical brushes with 2-isopropyloxazoline side chains close to the phase boundary.

A high-molar-mass cylindrical brush polymer with a main chain degree of polymerization of Pw = 1047 is synthesized by free-radical polymerization of a poly-2-isopropyloxazoline macromonomer with Pn = 28. The polymerization is conducted above the lower phase transition temperature of the macromonomer, i.e., in the phase-separated regime, which provides a sufficiently concentrated macromonomer phase mandatory to obtain high-molar-mass cylindrical brushes. Upon heating to the phase transition temperature, the hydrodynamic radius is observed to shrink from 34 to 27 nm. Further increase in temperature resulted in aggregated chains which were observed to coexist with single chains until eventually only aggregates of µm size were detectable.