Levosimendan increases brain tissue oxygen levels after cardiopulmonary resuscitation independent of cardiac function and cerebral perfusion.

Prompt reperfusion is important to rescue ischemic tissue; however, the process itself presents a key pathomechanism that contributes to a poor outcome following cardiac arrest. Experimental data have suggested the use of levosimendan to limit ischemia-reperfusion injury by improving cerebral microcirculation. However, recent studies have questioned this effect. The present study aimed to investigate the influence on hemodynamic parameters, cerebral perfusion and oxygenation following cardiac arrest by ventricular fibrillation in juvenile male pigs. Following the return of spontaneous circulation (ROSC), animals were randomly assigned to levosimendan (12 µg/kg, followed by 0.3 µg/kg/min) or vehicle treatment for 6 h. Levosimendan-treated animals showed significantly higher brain PbtO2 levels. This effect was not accompanied by changes in cardiac output, preload and afterload, arterial blood pressure, or cerebral microcirculation indicating a local effect. Cerebral oxygenation is key to minimizing damage, and thus, current concepts are aimed at improving impaired cardiac output or cerebral perfusion. In the present study, we showed that NIRS does not reliably detect low PbtO2 levels and that levosimendan increases brain oxygen content. Thus, levosimendan may present a promising therapeutic approach to rescue brain tissue at risk following cardiac arrest or ischemic events such as stroke or traumatic brain injury.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

A novel bovine papillomavirus type in the genus Dyokappapapillomavirus.

Papillomaviruses are a diverse group of viruses that are known to infect a wide range of animal species. Bovine papillomaviruses (BPVs) are divided into at least 21 genotypes (BPV1 to BPV21),  with most BPV isolates/strains described to date belonging to one of four genera, including Deltapapillomavirus, Xipapillomavirus, Epsilonpapillomavirus and Dyoxipapillomavirus. Here, we describe the identification and genetic characterization of a new BPV type in the genus Dyokappapapillomavirus. A farm in the state of New York, USA, reported chronic cases of vulvovaginitis in Holstein cows in 2016. Biopsies and/or swab samples collected from the vaginal mucosa were subjected to diagnostic investigation. Conventional diagnostic assays yielded negative results, and vaginal swab samples were subjected to viral metagenomic sequencing. Notably, BLAST searches revealed a papillomavirus genome with 7480 bp in length (67% nt sequence identity to BPV16). Additionally, phylogenetic analysis of the L1 gene of the papillomavirus identified here (tentatively named BPV22) revealed that it clusters with members of the genus Dyokappapapillomavirus. Interestingly, the recently identified BPV16, which was detected in fibropapilloma lesions in cattle also clusters within the Dyokappapapillomavirus group. Each virus, however, forms a separate branch in the phylogenetic tree. These results indicate that the putative BPV22 represents the second BPV within the genus Dyokappapapillomavirus.

Small Surfactant Concentration Differences Influence Adsorption of Human Serum Albumin on Polystyrene Nanoparticles.

Surfactants, even in miniscule amounts, are often used for the synthesis and especially the stabilization of nanomaterials, which is essential for in vivo applications. In this study, we show that the interaction between nanoparticles and proteins strongly depends on the type of stabilizing surfactants and their (small) concentration changes. The reaction between human serum albumin and polystyrene nanoparticles stabilized by an ionic or nonionic surfactant-sodium dodecyl sulfate or Lutensol AT50, respectively-was monitored using isothermal titration calorimetry. It was found that the amount of surfactant molecules on the surface significantly determines the protein binding affinity and adsorption stoichiometry, which is important for all nanomaterials coming into contact with biological components such as blood plasma proteins. Thus after synthesizing nanomaterials for in vivo applications as drug delivery agents, it is crucial to perform a detailed analysis of the obtained surface chemistry that accounts for the presence of minimal amounts of stabilizing agents.

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.

Fluorescence labels may significantly affect the protein adsorption on hydrophilic nanomaterials.

Fluorescently labelled proteins are often used to study processes in vitro, e.g. the binding of proteins to cell surfaces or the adsorption of plasma proteins on drug nanocarriers. However, the fact that the fluorescent labelling may affect the protein properties is frequently neglected. On the example of a simple model system, we reiterate the importance of this issue by showing that even a single label may perturb interactions between hydrophilic starch-based nanocapsules and serum albumin and thus prevent binding.

Osmotic pressure-dependent release profiles of payloads from nanocontainers by co-encapsulation of simple salts.

The encapsulation of payloads in micro- to nano-scale capsules allows protection of the payload from the surrounding environment and control of its release profile. Herein, we program the release of hydrophilic payloads from nanocontainers by co-encapsulating simple inorganic salts for adjusting the osmotic pressure. The latter either leads to a burst release at high concentrations of co-encapsulated salts or a sustained release at lower concentrations. Osmotic pressure causes swelling of the nanocapsule's shell and therefore sustained release profiles can be adjusted by crosslinking it. The approach presented allows for programing the release of payloads by co-encapsulating inexpensive salts inside nanocontainers without the help of stimuli-responsive materials.

Polycarbonate and polystyrene nanoplastic particles act as stressors to the innate immune system of fathead minnow (Pimephales promelas).

Water pollution with large-scale and small-scale plastic litter is an area of growing concern. Macro-plastic litter is a well-known threat to aquatic wildlife; however, the effects of micro-sized and nano-sized plastic particles on the health of organisms are not well understood. Small-scale plastic particles can easily be ingested by various aquatic organisms and potentially interfere with their immune system; therefore, the authors used a freshwater fish species as a model organism for nanoplastic exposure. Characterization of polystyrene (41.0 nm) and polycarbonate (158.7 nm) nanoplastic particles (PSNPs and PCNPs, respectively) in plasma was performed, and the effects of PSNPs and PCNPs on the innate immune system of fathead minnow were investigated. In vitro effects of PSNPs and PCNPs on neutrophil function were determined using a battery of neutrophil function assays. Exposure of neutrophils to PSNPs or PCNPs caused significant increases in degranulation of primary granules and neutrophil extracellular trap release compared to a nontreated control, whereas oxidative burst was less affected. The present study outlines the stress response of the cellular component of fish innate immune system to polystyrene and polycarbonate nanoparticles/aggregates and indicates their potential to interfere with disease resistance in fish populations. Environ Toxicol Chem 2016;35:3093-3100. © 2016 SETAC.

Detection of the Emerging Picornavirus Senecavirus A in Pigs, Mice, and Houseflies.

Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.

Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers.

The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.

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.

Nanocarrier for Oral Peptide Delivery Produced by Polyelectrolyte Complexation in Nanoconfinement.

The hydrophilic peptide YY (PYY) is a promising hormone-based antiobesity drug. We present a new concept for the delivery of PYY from pH-responsive chitosan-based nanocarriers. To overcome the drawbacks while retaining the merits of the polyelectrolyte complex (PEC) method, we propose a one-pot approach for the encapsulation of a hydrophilic peptide drug in cross-linked PEC nanocarriers. First, the hydrophilic peptide is encapsulated via polyelectrolyte complexation within water-in-oil miniemulsion droplets. In a second step, the PEC surface is reinforced by controlled interfacial cross-linking. PYY is efficiently encapsulated and released upon pH change. Such nanocarriers are promising candidates for the fight against obesity and, in general, for the oral delivery of protein drugs.

Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona.

Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so-called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES-NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of "stealth" and targeting behavior.

Biodegradable protein nanocontainers.

The application of synthetic polymers for drug delivery often requires tremendous efforts to ensure biocompatibility and -degradation. To use the body's own substances can help to overcome these problems. Herein, we present the first synthesis of nanocontainers entirely composed of albumin proteins. These protein nanocontainers (PNCs) were loaded with hydrophilic compounds and release of the payload is triggered through natural lysis in vitro in human monocyte-derived dendritic cells (moDCs). No aggregation of PNCs in human blood plasma was observed, indicating stability for blood circulation. As the PNCs were readily taken up by moDCs, they are considered as a promising delivery platform for vaccination strategies and could minimize the risk of side effects caused by foreign carrier substances.

Evaluation of multifunctional liposomes in human blood serum by light scattering.

To overcome the limited functionality of "stealth" lipids based on linear poly(ethylene glycol) (PEG) chains, hyperbranched polyether-based lipids that bear multiple hydroxyl groups for further chemical modification may be a suitable replacement. This study focuses on the development and characterization of "stealth" liposomes modified with a novel hyperbranched polyglycerol lipid (cholesterol-PEG30-hbPG23). An emphasis was placed on the stability of these liposomes in comparison to those containing a linear PEG derivative (cholesterol-PEG44) directly in human blood serum, characterized via dynamic light scattering (DLS). Polymer lipid contents were varied between 0 and 30 mol %, resulting in liposomes with sizes between 150 and 80 nm in radius, depending on the composition. DLS analysis showed no aggregation inducing interactions between serum components and liposomes containing 10-30 mol % of the hyperbranched lipid. In contrast, liposomes functionalized with comparable amounts of linear PEG exhibited aggregate formation in the size range of 170-330 nm under similar conditions. In addition to DLS, cryo-transmission electron microscopy (TEM) was employed for all liposome samples to prove the formation of unilamellar vesicles. These results demonstrate the outstanding potential of the introduction of hyperbranched polyglycerol into liposomes to stabilize the assemblies against aggregation while providing additional functionalization sites.

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.

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

HPMA-LMA copolymer drug carriers in oncology: an in vivo PET study to assess the tumor line-specific polymer uptake and body distribution.

Polymeric drug carriers aim to selectively target tumors in combination with protecting normal tissue. In this regard polymer structure and molecular weight are key factors considering organ distribution and tumor accumulation of the polymeric drug delivery system. Four different HPMA based copolymer structures (random as well as block copolymers with lauryl methacrylate as hydrophobic block) varying in molecular weight, size and resulting architecture were analyzed in two different tumor models (AT1 prostate carcinoma and Walker-256 mammary carcinoma) in vivo. Polymers were labeled with (18)F and organ/tumor uptake was followed by µPET imaging and ex vivo biodistribution. Vascular permeability was measured by dextran extravasation and vascular density by immunohistochemistry. Cellular polymer uptake was determined in vitro using fluorescence-labeled polymers. Most strikingly, the high molecular weight HPMA-LMA random copolymer demonstrated highest tumor uptake and blood pool concentration. The molecular structure (e.g., amphiphilicity) is holding a higher impact on desired in vivo properties than polymer size. The results also revealed pronounced differences between the tumor models although vascular permeability was almost comparable. Accumulation in Walker-256 carcinomas was much higher, presumably due to a better cellular uptake in this cell line and a denser vascular network in the tumors. These investigations clearly indicate that the properties of the individual tumor determine the suitability of polymeric drug carriers. The findings also illustrate the general necessity of a preclinical screening to analyze polymer uptake for each individual patient (e.g., by noninvasive PET imaging) in order to individualize polymer-based chemotherapy.