Search

A passive cavitation detection setup is used to assess and verify the capability of frequency- and amplitudemodulated ultrasound burst signals on the generation of inertial cavitation. The measurements were conducted with a flowthrough tissue-mimicking phantom with a canal of dc = 1mm in diameter simulating a fine blood vessel. By means of a flow velocity of vf= 50mm/s circulating blood in the cardiovascular system is imitated. Applying signal frequencies from f = 550 − 950 kHz and peak rarefaction pressures in a range from ˆpPRFP ≈ 0.17 − 1.81 MPa, various ultrasound stimuli with alternating signal parameters were investigated in the absence and presence of a talcum-water mixture acting as cavitation nuclei. By evaluating the broadband emissions in the frequency domain and calculating the voltage spectral density Srm, the results can be compared. The findings of the present study demonstrate that each ultrasound stimulus triggers a similar strength of cavitation noise at the same Mechanical Index, representing a certain pressure. Over the whole frequency and Mechanical Index (pressure) range, each ultrasound signal type generates a voltage spectral density from Srm= 0.25 − 2.25 V/ √ Hz.

Magnetic nanoparticles (MNPs) are interesting for various applications in medicine. If administered to a biological system like the human body, a so-called biomolecule corona is formed on the surface of the particles, which highly determines the biological fate of the particles. To elucidate whether a preconditioning of the MNPs by incubation with biomolecules influences biocompatibility and bioavailability, the formation of such a corona was investigated in more detail. For this, the influence of particle characteristics, e.g., surface charge, as well as various incubation parameters on the resulting corona was investigated. It was found that the biomolecule corona is formed immediately after bringing together the particles with the biomolecule source. By variation of the biomolecule content of the incubation medium, the size of the corona can be modulated. Regarding the interaction of the nanoparticles with cells, it was shown that the presence of a biomolecule corona reduces the interaction and that a more pronounced biomolecule corona leads to a reduced uptake of the magnetic nanohybrids into the cells. Cell viability tests confirmed biocompatibility of the biomolecule-coated particles. A more pronounced corona promotes a higher cell viability. By using a shell-less hen’s egg model, no or reduced adverse effects of all biomolecule-coated MNP for this in vivo test were found. Resulting from these investigations, we were able to demonstrate that our newly developed nanohybrids significantly reduce in vivo toxicity compared to uncoated MNPs.

Background: Poly(lactic-co-glycolic) acid (PLGA) nanoparticles can be prepared by emulsion-solvent-evaporation from o/w and w1/o/w2 emulsions. Aims: To elaborate similarities and differences regarding mechanical, morphological and physicochemical properties, as well as endocytosis and dose-dependent immune responses by primary human leukocytes between nanoparticles prepared by these two methods. Methods: Fluorescently labeled as well as TLR agonist (R848)-loaded PLGA nanoparticles were prepared via both single- and double-emulsion solvent evaporation. Results: Particles prepared by both methods were similar in chemical composition and surface charge but exhibited slight differences in size and morphology. Pronounced differences were found for loading, dissolution and mechanical properties. The particles were differently endocytosed by monocytes and induced qualitatively and quantitatively different immune responses. Conclusions: Variations in nanoparticle preparation can affect particle-derived immunological characteristics.

Simple Summary Hepatocellular carcinoma (HCC) ranks second among the leading causes of cancer-related death. Since current therapeutic options are very limited, a deeper understanding of the molecular mechanisms underlying the tumor onset and progression of HCC holds great potential for improved therapeutic options. Although it has been shown that deleted in liver cancer 1 (DLC1) acts as a tumor suppressor whose allele is lost in 50% of liver cancers, alterations in gene expression initiated by DLC1 loss have not yet been the primary focus of liver cancer research. To identify novel gene targets that allow for a personalized medicine approach for HCC therapy, we performed gene expression profiling for HepG2 cells stably expressing DLC1shRNA. We provide evidence that TSPAN5 is required for HCC growth, migration and invasion, and dissected the underlying molecular mechanisms involving myocardin-related transcription factors. Thus, TSPAN5 represents a novel therapeutic target for the treatment of HCC characterized by DLC1 loss. Abstract Human hepatocellular carcinoma (HCC) is among the most lethal and common cancers in the human population, and new molecular targets for therapeutic intervention are urgently needed. Deleted in liver cancer 1 (DLC1) was originally identified as a tumor suppressor gene in human HCC. DLC1 is a Rho-GTPase-activating protein (RhoGAP) which accelerates the return of RhoGTPases to an inactive state. We recently described that the restoration of DLC1 expression induces cellular senescence. However, this principle is not amenable to direct therapeutic targeting. We therefore performed gene expression profiling for HepG2 cells depleted of DLC1 to identify druggable gene targets mediating the effects of DLC1 on senescence induction. This approach revealed that versican (VCAN), tetraspanin 5 (TSPAN5) and N-cadherin (CDH2) were strongly upregulated upon DLC1 depletion in HCC cells, but only TSPAN5 affected the proliferation of HCC cells and human HCC. The depletion of TSPAN5 induced oncogene-induced senescence (OIS), mediated by the p16INK4a/pRb pathways. Mechanistically, silencing TSPAN5 reduced actin polymerization and thereby myocardin-related transcription factor A- filamin A (MRTF-A-FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction in vitro and in vivo. Our results identify TSPAN5 as a novel druggable target for HCC.

Tripterygium wilfordii glycosides (TWG) is a traditional Chinese medicine with effectiveness against rheumatoid arthritis (RA), supported by numerous clinical trials. Lipid mediators (LM) are biomolecules produced from polyunsaturated fatty acids mainly by cyclooxygenases (COX) and lipoxygenases (LOX) in complex networks which regulate inflammation and immune responses and are strongly linked to RA. The mechanism by which TWG affects LM networks in RA treatment remains elusive. Employing LM metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry revealed striking modulation of LM pathways by TWG in human monocyte-derived macrophage (MDM) phenotypes. In inflammatory M1-MDM, TWG (30 µg/mL) potently suppressed agonist-induced formation of 5-LOX products which was confirmed in human PMNL and traced back to direct inhibition of 5-LOX (IC50 = 2.9 µg/mL). TWG also efficiently blocked thromboxane formation in M1-MDM without inhibiting other prostanoids and COX enzymes. Importantly, in anti-inflammatory M2-MDM, TWG (30 µg/mL) induced pronounced formation of specialized pro-resolving mediators (SPM) and related 12/15-LOX-derived SPM precursors, without COX and 5-LOX activation. During MDM polarization, TWG (1 µg/mL) decreased the capacity to generate pro-inflammatory 5-LOX and COX products, cytokines and markers for M1 phenotypes. Together, suppression of pro-inflammatory LM but SPM induction may contribute to the antirheumatic properties of TWG.

Aim: To simulate the stability and degradation of superparamagnetic iron oxide nanoparticles (MNP) in vitro as part of their life cycle using complex simulated biological fluids. Materials & methods: A set of 13 MNP with different polymeric or inorganic shell materials was synthesized and characterized regarding stability and degradation of core and shell in simulated biological fluids. Results: All MNP formulations showed excellent stability during storage and in simulated body fluid. In endosomal/lysosomal media the degradation behavior depended on shell characteristics (e.g., charge, acid-base character) and temperature enabling the development of an accelerated stress test protocol. Conclusion: Kinetics of transformations depending on the MNP type could be established to define structure-activity relationships as prediction model for rational particle design.

Magnetic nanocomposites are a class of smart materials that have attracted recent interest as drug delivery systems or as medical implants. In this study, meltable nanobiocomposites (NBC) composed of biocompatible dextran fatty acid ester and magnetic nanoparticles (MNPs) melting close to human body temperature were prepared and loaded with Rhodamine B (RhB) or green fluorescent protein (GFP) as model drugs to evaluate their potential use as drug delivery system. The release of the model drugs from the magnetic NBC investigated under the influence of a high frequent alternating magnetic field (AMF, 20 kA/m at 400 kHz) showed that on-demand release is realized applying the external AMF. The NBC showed a long-term stability (28 d) of the incorporated iron oxide particles after incubation in artificial body fluids. This work reveals the potential of the NBC as a drug carrier.

Natural products suited for prophylaxis and therapy of inflammatory diseases have gained increasing importance. These compounds could be beneficially integrated into bacterial cellulose (BC), which is a natural hydropolymer applicable as a wound dressing and drug delivery system alike. This study presents experimental outcomes for a natural anti-inflammatory product concept of boswellic acids from frankincense formulated in BC. Using esterification respectively (resp.) oxidation and subsequent coupling with phenylalanine and tryptophan, post-modification of BC was tested to facilitate lipophilic active pharmaceutical ingredient (API) incorporation. Diclofenac sodium and indomethacin were used as anti-inflammatory model drugs before the findings were transferred to boswellic acids. By acetylation of BC fibers, the loading efficiency for the more lipophilic API indomethacin and the release was increased by up to 65.6% and 25%, respectively, while no significant differences in loading could be found for the API diclofenac sodium. Post-modifications could be made while preserving biocompatibility, essential wound dressing properties and anti-inflammatory efficacy. Eventually, in vitro wound closure experiments and evaluations of the effect of secondary dressings completed the study.

This report highlights the importance of hydrophobic groups mimicking the side chains of aromatic amino acids, which are tryptophan, phenylalanine, and tyrosine, in guanidinium bearing poly(methacrylamide)s for the design of non-viral gene delivery agents. Guanidinium containing methacrylamide terpolymers are prepared by aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization with different hydrophobic monomers, N-(2-indolethyl)methacrylamide (IEMA), N-phenethylmethacrylamide (PhEMA), or N-(4-hydroxyphenethyl)methacrylamide (PhOHEMA) by aiming similar contents. The well-defined polymers are obtained with a molar mass of ≈15 000 g mol-1 and ≈1.1 dispersity. All terpolymers demonstrate almost comparable in vitro cell viability and hemocompatibility profiles independent of the type of side chain. Although they all form positively charged, enzymatically stable polyplexes with plasmid DNA smaller than 200 nm, the incorporation of the IEMA monomer improve these parameters by demonstrating a higher DNA binding affinity and forming nanoassemblies of about 100 nm. These physicochemical characteristics are correlated with increased transfection rates in CHO-K1 cells dependent on the type of the monomer and the nitrogen to phosphate (N/P) ratio of the polyplexes, as determined by luciferase reporter gene assays.

Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such asMortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products ofMortierella alpinaATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey’s method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tetheredl-amino acids and the subsequent incorporation ofd-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. This is the first report of nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products.IMPORTANCEFungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genusMortierellaas representative of the early diverging fungi as an underestimated resource of natural products.Mortierella alpinaproduces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes inMortierella. Both enzymes are the first biochemically characterized natural product biosynthesis enzymes of basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

Dextran-formamidine esters (dextran-N-[(dimethylamino)methylene]-β-alanine ester) with different degrees of substitution (0.45-0.92) are synthesized in an one-pot reaction. Dextran (Mw 60 000 g mol-1 ) is allowed to react with unprotected beta-alanine and iminium chloride and investigated regarding the potential as gene delivery system for the transfer of plasmid DNA. With degrees of substitution ≥ 0.63 improved DNA binding with formation of enzymatically stable complexes of about 130-160 nm with negative surface charges are obtained. These physicochemical characteristics correlated with increasing transfection rates in CHO-K1 cells determined by a luciferase reporter gene assay in dependency of the number of formamidine residues, N/P ratios and amount of DNA. The role of the number of formamidine groups is also highlighted by in vitro cyto- and hemotoxicity tests under the chosen conditions. These results indicate that dextran-formamidine esters are a very promising material for the safe and efficient gene delivery.

Bacterial nanocellulose (BNC) is a highly interesting biomaterial due to some outstanding properties especially when used in medical therapeutics and diagnostics. BNC is absolutely bioinert and is characterised by intrinsic properties such as high tensile stiffness and elasticity, high porosity, exceptional water uptake and swelling capacity. Furthermore, these properties can be adjusted in a very defined way by specifically changing the cultivation conditions or performing post-modifications such as crosslinking, functionalisation with additives, dehydration or drying. Especially the high tensile strength of the nanofibrillar material has been the subject of many investigations in the past couple of years. Nevertheless, the enormous tensile strength and elasticity of BNC is contrary to an almost purely viscous behaviour under compressive load. In the present study, different methods to influence the mechanical behaviour under compression with respect to load bearing applications of BNC are systematically investigated. The possibilities and limitations of the variable layer-by-layer cultivation known as Mobile Matrix Reservoir Technology (MMR-Tech) as well as the effect of different post-modification strategies of BNC are thoroughly investigated. Beside of commonly used indentation tests for characterising the mechanical properties of BNC, we introduce a novel evaluation methodology based on mechanical relaxation measurements and an evolutionary regression algorithm for the derivation of a viscoelastic material law, which for the first time allows standardised, comparative viscoelastic investigations of soft-matter biomaterials to be performed independently of the measurement setup. Using this methodology, we are able to show, that cultivation conditions for BNC and suitable post-modifications can result in different effects on the viscoelastic behaviour of the fabricated composites. We show that the cultivation conditions for BNC primarily affect the height of dispersion and the frequency of the relaxation centre which corresponds roughly to the mean value of the logarithmic distributed relaxation times, and that these effects could be enhanced by post-modifications. However, we also identify parameters, such as the width of the relaxation region, which corresponds roughly to the standard deviation of the logarithmic distributed relaxation times, on which the type of cultivation obviously shows no influence but which can be influenced exclusively by post-modifications. Our methodology enables for the first time a clear identification of those parameters which represent a significant factor of influence to the viscoelastic material behaviour, which should enable a more targeted and application-relevant development of BNC composites in the future.

This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).

IntroductionHealthcare-associated infections (HAIs) are a major health concern and have substantial effects on morbidity and mortality and increase healthcare costs. We investigated the effect of a hospital-wide program for the prevention of HAIs on additional length of stay (LOS).MethodsWe analyzed data from a prospective, single-center, quasi-experimental study with two surveillance periods before and after implementation of an infection prevention intervention program. HAI diagnosis was made according to surveillance definition criteria established by the US Centers for Disease Control and Prevention. A multistate model was used to estimate additional LOS for patients with HAI in both surveillance periods.ResultsDuring the first and second periods, 1,568 and 2,336 HAIs were identified among 26,943 and 35,211 patients, respectively. For HAI patients exclusively treated in a general ward, additional LOS was 8.4 (95% confidence interval, CI: 6.8-10.0) days in the first period and 9.6 (95% CI: 8.3-11.0) days in the second period (p = 0.26). For HAI patients treated in both an intensive care unit (ICU) and a general ward, additional LOS was 8.1 (95% CI: 6.3-9.9) days in the first period to 7.3 (95% CI: 6.1-8.5) days in the second period (p = 0.47).ConclusionsHealthcare-associated infections prolong LOS. A hospital-wide infection control program did not alter the prolongation of LOS.

Mareike Klinger-Strobel,1,2,* Julia Ernst,3,* Christian Lautenschläger,4 Mathias W Pletz,1,2 Dagmar Fischer,3,5 Oliwia Makarewicz1,2 1Center for Infectious Diseases and Infection’s Control, 2Center for Sepsis Control and Care, Jena University Hospital, 3Department of Pharmaceutical Technology, Friedrich Schiller University Jena, 4Department of Internal Medicine IV, Jena University Hospital, 5Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Jena, Germany*These authors contributed equally tothis work Abstract: Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO® 9, propidium iodide, fluorescein). Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(D,L-lactide-co-glycolide) (PLGA)-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA), after which blue fluorescent poly(ethylene glycol)-block-PLGA (PEG-PLGA) particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. Keywords: 7-amino-4-methyl-3-coumarinylacetic acid, PLGA, PEG, confocal laser scanning microscopy, cystic fibrosis, chitosan, hydrodynamic diameter

The histone deacetylase inhibitors (HDACi) are potent drugs in the treatment of inflammatory diseases and defined cancer types. However, major drawbacks of HDACi, such as valproic acid (VPA), are limited serum half-life, side effects and the short circulation time. Thus, the immobilization of VPA in a polysaccharide matrix is used to circumvent these problems and to design a suitable nanocarrier system. Therefore, VPA is covalently attached to cellulose and dextran via esterification with degree of substitution (DS) values of up to 2.20. The resulting hydrophobic polymers are shaped to spherical nanoparticles (NPs) with hydrodynamic diameter between 138 to 221 nm and polydispersity indices from 0.064 to 0.094 by nanoprecipitation and emulsification technique. Lipase treatment of the NPs leads to in vitro release of VPA and hence to an inhibition of HDAC2 activity in a HDAC2 assay. NPs are rapidly taken up by HeLa cells and mainly localize in the cytoplasm. The NPs are hemocompatible and nontoxic as revealed by the shell-less hen's egg model.

Nanocelluloses are natural materials with at least one dimension in the nano-scale. They combine important cellulose properties with the features of nanomaterials and open new horizons for materials science and its applications. The field of nanocellulose materials is subdivided into three domains: biotechnologically produced bacterial nanocellulose hydrogels, mechanically delaminated cellulose nanofibers, and hydrolytically extracted cellulose nanocrystals. This review article describes todays state regarding the production, structural details, physicochemical properties, and innovative applications of these nanocelluloses. Promising technical applications including gels/foams, thickeners/stabilizers as well as reinforcing agents have been proposed and research from last five years indicates new potential for groundbreaking innovations in the areas of cosmetic products, wound dressings, drug carriers, medical implants, tissue engineering, food and composites. The current state of worldwide commercialization and the challenge of reducing nanocellulose production costs are also discussed., Dana Kralisch and Dagmar Fischer gratefully acknowledge the Free State of Thuringia and the European Social Fund (2016 FGR 0045) for funding. Dagmar Fischer would like to thank Yvette Pötzinger and Berit Karl for the excellent editorial support. Dieter Klemm, Friederike Kramer and Katrin Petzold-Welcke are grateful for the support by the Federal Ministry of Economic Affairs and Energy, ZIM (KF2748903MF4 and KF2386003MF3). Thanks are due to the employees of Jenpolymer Materials Ltd. & Co. KG and the Polymet Jena Association, especially Priv.-Doz. Dr. Wolfgang Fried, and Prof. Dr. Raimund W. Kinne, Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital, Germany as well as to Dr. Detlef Gorski and Elke Langhammer, SuraChemicals GmbH, Jena, Germany for effective and helpful cooperation and stimulating interaction. Dieter Klemm and Friederike Kramer would like to thank Katharina Horn for the excellent editorial support. Miguel Gama acknowledges the funding from QREN (“Quadro de Referência Estratégica Nacional”) through the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte. Tom Lindström acknowledges RISE Bioeconomy for support and permission to publish. Emily Cranston and Stephanie Kedzior are thankful for funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) in the form of a Discovery Grant (RGPIN 402329) and PGSD graduate student scholarship, as well as support from the Faculty of Engineering at McMaster University., info:eu-repo/semantics/publishedVersion

When silica nanoparticles (SiNP) are stored in aqueous solution, even for few hours, they have a tendency to form agglomerates and therefore adapt inhomogeneous structures. Here we present a very practical method to store SiNP in responsive hydrogel. We have confirmed that SiNP kept in the responsive hydrogel do not undergo through undesirable morphological changes and while in storage they maintain their excellent colloidal stability. The effect of SiNP hollowing (i.e. dissolution of the core of the particles that leaves empty cavity inside) was significantly inhibited in the hydrogel, which is a critical feature for any nano-medical applications (e.g. controlled drug release). To demonstrate the applicability of the hydrogel-storing concept within a biologically relevant context, in this work we have evaluated the toxicological effects of the responsive SiNP-gel formulation in a model in vitro (human cell line U87GM and hemocompatibility using red blood cells) and ex ovo (hen's egg test) experiments. Particles stored in the gel as well as the pure gel did not affect the hemocompatibility (hemolysis and erythrocyte aggregation) up to a concentration of 100 µg/mL. Furthermore, systemic injections into the blood circulation of the chick area vasculosa confirmed the biocompatibility in a more complex biological environment. All evaluated toxicological values (hemorrhage, thrombosis, vascular lysis, and lethality) were comparable with the negative control, and no differences in toxicological response could be observed between the SiNP stored in hydrogel and the control nanoparticles stored in the solution.

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

Although bacterial nanocellulose (BNC), a natural nanostructured biopolymer network, offers unique material characteristics, the number of drug-loaded BNC-based carriers in clinical trials or on the market is still low. This report provides an overview of aspects still limiting the broad application of BNC as drug-delivery system and the challenges for its future applications. Continuous large-scale production, storability, the loading and controlled release of critical drugs, for example, with high molar mass or highly lipophilic character as well as the formulation of long-term release systems will be highlighted. Recent achievements toward promoting the application of BNC as drug-delivery system and overcoming these obstacles will be discussed. [Formula: see text]

In the steadily emerging field of applications for the natural biopolymer bacterial nanocellulose (BNC), the development of environmentally-friendly and cost-saving techniques to form xerogels by partial or complete dewatering is of great interest for convenient storage, handling and a reduced risk of microbial contamination. Air-dried BNC itself is not able to rehydrate after complete drying due to a structural collapse. In the present paper, it was supplemented with different hydrophilic, water-binding additives and characterized regarding morphology, re-swelling behavior, mechanical stability and potential as drug delivery system. A fast rehydration could be obtained by the addition of magnesium chloride > glucose > sucrose > sorbitol, with a maximum re-swelling percentage up to about 88% (magnesium chloride) of the initial wet weight. In contrast, poly(ethylene glycol) (8 kDa), lactose and trehalose reached only 12–30% whereas mannitol and sodium chloride only had a negligible effect. A high re-swelling value was found to be correlated with the preservation of the three-dimensional BNC network structure and mechanical characteristics such as compression and tensile strength. Confirming the relevance of these findings, the use of the hydrophilic model drug azorubine demonstrated the applicability of the shape-memorized bacterial nanocellulose as drug delivery system with controllable release profiles.

The aim of this study was to establish an in vitro model test system for the analysis of cellular effects of nanoparticles on endothelial cells using the human brain microvascular endothelial cell line HBMEC, a representative of the blood-brain barrier. At first we defined a toxic end point by using superparamagnetic iron oxide nanoparticles coated with cationic substances (polyethylenimine, chitosan, diethylamine ethyl). To study the toxic effects of nanoparticles we evaluated three different viability assays and one cytotoxicity assay for their suitability. We could show that a luminescence-based assay is most promising for an accurate testing, because it is reproducible and unaffected by assay conditions and reagents.

This article describes the preparation and the physico-chemical characterization of a new host-guest system consisting of zeolite beta nanoparticles as host and mitoxantrone as guest. The resulting host-guest system mitoxantrone@beta is characterized in terms of morphology (transmission electron microscopy, dynamic light scattering), structure (powder wide-angle X-ray diffraction, nitrogen sorption), surface charge (ξ-potential measurements), and optical properties (UV-visible absorption, steady-state fluorescence). Mitoxantrone@beta particles are monodisperse in size with a mean diameter centered around 100 nm. Mitoxantrone guest molecules are adsorbed at the micropore entrances of zeolite host. Resulting nanoparticles retrieve the interesting optical properties of guest molecules with a fluorescence emission band in the near-infrared region. Mitoxantrone loading is comparatively evaluated by three different means (elemental analysis, direct and indirect UV-visible absorption studies) showing a loading level of 6.8 μmol/g. Mitoxantrone@beta nanoparticles also show a noticeable cytotoxic effect when applied to cancer cells.

Bacterial nanocellulose (BNC) is chemically identical with plant cellulose but free of byproducts like lignin, pectin, and hemicelluloses, featuring a unique reticulate network of fine fibers. BNC sheets are mostly obtained by static cultivation. Now, a Horizontal Lift Reactor may provide a cost efficient method for mass production. This is of particular interest as BNC features several properties of an ideal wound dressing although it exhibits no bactericidal activity. Therefore, BNC was functionalized with the antiseptics povidone-iodine (PI) and polihexanide (PHMB). Drug loading and release, mechanical characteristics, biocompatibility, and antimicrobial efficacy were investigated. Antiseptics release was based on diffusion and swelling according to Ritger-Peppas equation. PI-loaded BNC demonstrated a delayed release compared to PHMB due to a high molar drug mass and structural changes induced by PI insertion into BNC that also increased the compressive strength of BNC samples. Biological assays demonstrated high biocompatibility of PI-loaded BNC in human keratinocytes but a distinctly lower antimicrobial activity against Staphylococcus aureus compared to PHMB-loaded BNC. Overall, BNC loaded with PHMB demonstrated a better therapeutic window. Moreover, compressive and tensile strength were not changed by incorporation of PHMB into BNC, and solidity during loading and release could be confirmed.

Biofilms are the preferred sessile and matrix-embedded life form of most microorganisms on surfaces. In the medical field, biofilms are a frequent cause of treatment failure because they protect the bacteria from antibiotics and immune cells. Antibiotics are selected according to the minimal inhibitory concentration (MIC) based on the planktonic form of bacteria. Determination of the minimal biofilm eradicating concentration (MBEC), which can be up to 1,000-fold greater than the MIC, is not currently conducted as routine diagnostic testing, primarily because of the methodical hurdles of available biofilm assessing protocols that are time- and cost-consuming. Comparative analysis of biofilms is also limited as most quantitative methods such as crystal violet staining are indirect and highly imprecise. In this paper, we present a novel algorithm for assessing biofilm resistance to antibiotics that overcomes several of the limitations of alternative methods. This algorithm aims for a computer-based analysis of confocal microscope 3D images of biofilms after live/dead stains providing various biofilm parameters such as numbers of viable and dead cells and their vertical distributions within the biofilm, or biofilm thickness. The performance of this algorithm was evaluated using computer-simulated 2D and 3D images of coccal and rodent cells varying different parameters such as cell density, shading or cell size. Finally, genuine biofilms that were untreated or treated with nitroxoline or colistin were analyzed and the results were compared with quantitative microbiological standard methods. This novel algorithm allows a direct, fast and reproducible analysis of biofilms after live/dead staining. It performed well in biofilms of moderate cell densities in a 2D set-up however the 3D analysis remains still imperfect and difficult to evaluate. Nevertheless, this is a first try to develop an easy but conclusive tool that eventually might be implemented into routine diagnostics to determine the MBEC and to improve outcomes of patients with biofilm-associated infections.

A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.

In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (

In somatic cells DNA topoisomerase II (topo II) is thought to be involved in the domain Organization of the genome by anchoring the basis of chromatin loops to a chromosomal scafFold. Lampbrush chromosomes of am-phibian oocytes directly display this radial loop Organization in cytological preparations. In order to find out whether topo II may play a role in the Organization of these meiotic chromosomes, we performed immunofluorescence studies using antibodies against Xenopus topo II. Our results indicate that topo II is apparently absent from lampbrush chromosomes and is hence unlikely to act as a "fastener" of the numerous lateral chromosomal loops. Topo II was, however, enriched in the amplified nucleoli of Xenopus oocytes.

To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1-4; DY-675, DY-676, DY-677, DY-678, respectively), were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection) using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the basis for the rationalization of future contrast agent developments.

Background: Before reunification, the post-neonatal mortality rate was lower in East Germany than in West Germany. Moreover, the incidence of SIDS (sudden infant death syndrome) was much lower in the East. Methods: Mortality data on sudden infant death syndrome (SIDS) from West and East Germany since 1980 as well as post-neonatal mortality data for both states since 1970 were examined. 95% Confidence intervals were calculated for the rates. Witnesses from the former East Germany who were involved at the time were also interviewed and archives were searched. Results: We found that as early as 1972 active monitoring of infant and child mortality rates in East Germany had shown that the prone sleeping position was dangerous for infants: the post-neonatal mortality rate was approximately 1 per 1000 live births lower in East than in West Germany during the 20 years before reunification. In contrast, in the West, prone sleeping was only discovered to be a risk factor for SIDS in the early 1990s. Conclusions: Active monitoring is an effective tool in the early detection of risk factors and serves to prevent unnecessary deaths.

The sequence-specific cleavage of RNA molecules through ribozyme targeting is particularly attractive since it allows the effective abrogation of protein expression. So far, however, use of enzymatically active RNA molecules (ribozymes) has, without chemical modification, been severely hampered by ribozyme instability and poor cellular uptake. In this paper, we present a method for protection and cellular delivery of ribozymes by complexation with a low molecular weight polyethylenimine (LMW-PEI). We show that LMW-PEI almost completely stabilizes ribozymes or any RNA against degradation in vitro. Upon their highly efficient cellular uptake, non-toxic LMW-PEI-complexed ribozymes display intracellular bioactivity already at low concentrations as demonstrated by down-regulation of two different genes in different cell lines. In vivo, LMW-PEI-complexed ribozymes were stabilized after intraperitoneal (i.p.) injections, showed prolonged circulation time and intact ribozymes were detected in the subcutaneous (s.c.) tumor mass 60 min after the injection. In addition, i.p. injections of LMW-PEI-complexed ribozymes targeted against the growth factor pleiotrophin (PTN) resulted in marked reduction of s.c. human melanoma tumor growth and of intratumoral PTN levels in a mouse xenograft model. Thus, this paper describes a novel method for exogenous delivery of any bioactive RNA ribozyme in vitro and in vivo without chemical modification.

Endoglin is a component of the transforming growth factor-β receptor complex whose expression is limited to a small number of cell types, including endothelial cells (ECs), activated monocytes, tissue macrophages and erythroid precursors. Of particular interest is its preferential expression in the vasculature of many malignant tumors, especially in view of potential therapeutic applications. We have cloned the human endoglin promoter, analyzed its structure and demonstrate that the isolated genomic fragment shows strong promoter activity in ECs (compared to other known EC-selective promoters), but not in epithelial cells and fibroblasts. These findings suggest that the endoglin promoter may prove a useful tool for the transcriptional targeting of ECs by gene therapy. We also determined the domains that are responsible for both efficient transcription and the observed preferential activity in ECs. The region around the major site of transcription initiation was found to be essential for transcription in both ECs and non-ECs. In contrast, cell type specificity does not appear to be governed by a single mechanism, but rather seems to be due to functionally distinct regulatory mechanisms acting on different upstream sequences.

Available data on the occurrence and expression of somatic histone H1 during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone H1A in oocytes is difficult to reconcile with the high transcriptional activity of all gene classes in this specific cell type. In the present study we have used polyclonal antibodies raised against somatic Xenopus histone H1 (H1A and H1A/B) for combined immunoblotting experiments to quantitate H1 pools and immunolocalization studies to visualize chromosome-bound H1. Both approaches failed to detect soluble or chromosomal histone H1 in vitellogenic oocytes, eggs, and cleavage-stage embryos up to early blastula. In addition, chromatin assembled in Xenopus egg extract was also negative for histone H1 as revealed by immunofluorescence microscopy. Lampbrush chromosomes not only lacked histone H1 but also the previously identified histone H1-like B4 protein (Smith et al., 1988, Genes Dev. 2, 1284-1295). In contrast, chromosomes of eggs and early embryos fluoresced brightly with anti-B4 antibodies. Our results lend further support to the view that historic H1 expression is developmentally regulated during Xenopus oogenesis and embryogenesis similar to what is known from other species.

Nucleoli provide the fascinating possibility of linking mor- phologically distinct structures such as those seen in the electron microscope with biochemical features of the for- mation and stepwise maturation of ribosomes. Localiza- tion of proteins by immunocytochemistry and of rRNA genes and their transcripts by in situ hybridization has greatly improved our understanding of the structural-func- tional relationships of the nucleolus. The present review describes some recent results obtained by electron micro- scopic in situ hybridization and argues that this approach has the potential to correlate each step of the complex pre-rRNA maturation pathway with nucleolar structures. Evidence is accumulating that the nucleolus-specific U3 snRNPs (small nuclear ribonucleoprotein particles) parti- cipate in rRNA processing events, similar to the role played by the nucleoplasmic snRNPs in mRNA matura- tion. The intranucleolar distribution of U3 snRNA is con- sistent with the view that it is involved in both early and late stages of pre-rRNA processing.