Inhibition of IL-1ß release from macrophages targeted with necrosulfonamide-loaded porous nanoparticles.

Inflammation is required for protective responses against pathogens and is thus essential for survival, but sustained inflammation can lead to diseases, such as atherosclerosis and cancer. Two important mediators of inflammation are the cytokines IL-1ß and IL-18, which are produced by myeloid cells of the immune system, including macrophages. These cytokines are released into the extracellular space through pores formed in the plasma membrane by the oligomerized protein gasdermin D (GSDMD). Necrosulfonamide (NSA) was recently identified as an effective GSDMD inhibitor and represents a promising therapeutic agent in GSDMD-dependent inflammatory diseases. Here, we targeted NSA to both mouse and human macrophages by using three different types of porous nanoparticles (NP), i.e. mesoporous silica (MSN), porous crosslinked cyclodextrin carriers (CD-NP), and a mesoporous magnesium-phosphate carrier (MPC-NP), all displaying high loading capacities for this hydrophobic drug. Cellular uptake and intracellular NSA delivery were tracked in time-lapse experiments by live-cell, high-throughput fluorescence microscopy, demonstrating rapid nanoparticle uptake and effective targeted delivery of NSA to phagocytic cells. Notably, a strong cytostatic effect was observed when a macrophage cell line was exposed to free NSA. In contrast, cell growth was much less affected when NSA was delivered via the nanoparticle carriers. Utilizing NSA-loaded nanoparticles, a successful concentration-dependent suppression of IL-1ß secretion from freshly differentiated primary murine and human macrophages was observed. Functional assays showed the strongest suppressive effect on human macrophages when using CD-NP for NSA delivery, followed by MSN-NP. In contrast, MPC-NP completely blocked the metabolic activity in macrophages when loaded with NSA. This study demonstrates the potential of porous nanoparticles for the effective delivery of hydrophobic drugs to macrophages in order to suppress inflammatory responses.

Curcumin Encapsulated in Crosslinked Cyclodextrin Nanoparticles Enables Immediate Inhibition of Cell Growth and Efficient Killing of Cancer Cells.

The efficiency of anti-cancer drugs is commonly determined by endpoint assays after extended incubation times, often after days. Here we demonstrate that curcumin encapsulated in crosslinked cyclodextrin nanoparticles (CD-NP) acts extremely rapidly on cell metabolism resulting in an immediate and complete inhibition of cell growth and in efficient cancer-cell killing only few hours after incubation. This early onset of anti-cancer action was discovered by live-cell high-throughput fluorescence microscopy using an environmental stage. To date, only very few examples of covalently crosslinked nanoscale CD-based (CD-NP) drug carriers exist. Crosslinking cyclodextrins enables the adsorption of unusually high payloads of hydrophobic curcumin (762 µg CC/mg CD-NP) reflecting a molar ratio of 2.3:1 curcumin to cyclodextrin. We have investigated the effect of CD-NP encapsulated curcumin (CD-CC-NP) in comparison to free, DMSO-derived curcumin nanoparticles (CC-NP) on 4 different cell lines. Very short incubations times as low as 1 h were applied and cell responses after medium change were subsequently followed over two days. We show that cell proliferation is inhibited nearly immediately in all cell lines and that a cell- and concentration dependent cancer-cell killing occurs. Anti-cancer effects were similar with free and encapsulated curcumin, however, encapsulation in CD-NP drastically extends the long-term photostability and anti-cancer activity of curcumin. Curcumin-sensitivity is highest in HeLa cells reaching up to 90% cell death under these conditions. Sensitivity decreased from HeLa to T24 to MDA MB-231 cells. Strikingly, the immortalized non-cancerous cell line MCF-10A was robust against curcumin concentrations that were highly toxic to the other cell lines. Our results underline the potential of curcumin as gentle and yet effective natural anti-cancer agent when delivered solvent-free in stabilizing and biocompatible drug carriers such as CD-NP that enable efficient cellular delivery.

Particle-Size-Dependent Delivery of Antitumoral miRNA Using Targeted Mesoporous Silica Nanoparticles.

Multifunctional core-shell mesoporous silica nanoparticles (MSN) were tailored in size ranging from 60 to 160 nm as delivery agents for antitumoral microRNA (miRNA). The positively charged particle core with a pore diameter of about 5 nm and a stellate pore morphology allowed for an internal, protective adsorption of the fragile miRNA cargo. A negatively charged particle surface enabled the association of a deliberately designed block copolymer with the MSN shell by charge-matching, simultaneously acting as a capping as well as endosomal release agent. Furthermore, the copolymer was functionalized with the peptide ligand GE11 targeting the epidermal growth factor receptor, EGFR. These multifunctional nanoparticles showed an enhanced uptake into EGFR-overexpressing T24 bladder cancer cells through receptor-mediated cellular internalization. A luciferase gene knock-down of up to 65% and additional antitumoral effects such as a decreased cell migration as well as changes in cell cycle were observed. We demonstrate that nanoparticles with a diameter of 160 nm show the fastest cellular internalization after a very short incubation time of 45 min and produce the highest level of gene knock-down.

Local emergency medical response after a terrorist attack in Norway: a qualitative study.

INTRODUCTION: On 22 July 2011, Norway suffered a devastating terrorist attack targeting a political youth camp on a remote island. Within a few hours, 35 injured terrorist victims were admitted to the local Ringerike community hospital. All victims survived. The local emergency medical service (EMS), despite limited resources, was evaluated by three external bodies as successful in handling this crisis. This study investigates the determinants for the success of that EMS as a model for quality improvement in healthcare. METHODS: We performed focus group interviews using the critical incident technique with 30 healthcare professionals involved in the care of the attack victims to establish determinants of the EMS' success. Two independent teams of professional experts classified and validated the identified determinants. RESULTS: Our findings suggest a combination of four elements essential for the success of the EMS: (1) major emergency preparedness and competence based on continuous planning, training and learning; (2) crisis management based on knowledge, trust and data collection; (3) empowerment through multiprofessional networks; and (4) the ability to improvise based on acquired structure and competence. The informants reported the successful response was specifically based on multiprofessional trauma education, team training, and prehospital and in-hospital networking including mental healthcare. The powerful combination of preparedness, competence and crisis management built on empowerment enabled the healthcare workers to trust themselves and each other to make professional decisions and creative improvisations in an unpredictable situation. CONCLUSION: The determinants for success derived from this qualitative study (preparedness, management, networking, ability to improvise) may be universally applicable to understanding the conditions for resilient and safe healthcare services, and of general interest for quality improvement in healthcare.

Neuronal distribution of tyramine and the tyramine receptor AmTAR1 in the honeybee brain.

Tyramine is an important neurotransmitter, neuromodulator, and neurohormone in insects. In honeybees, it is assumed to have functions in modulating sensory responsiveness and controlling motor behavior. Tyramine can bind to two characterized receptors in honeybees, both of which are coupled to intracellular cAMP pathways. How tyramine acts on neuronal, cellular and circuit levels is unclear. We investigated the spatial brain expression of the tyramine receptor AmTAR1 using a specific antibody. This antibody detects a membrane protein of the expected molecular weight in western blot analysis. In honeybee brains, it labels different structures which process sensory information. Labeling along the antennal nerve, in projections of the dorsal lobe and in the gnathal ganglion suggest that tyramine receptors are involved in modulating gustatory and tactile perception. Furthermore, the ellipsoid body of the central complex and giant synapses in the lateral complex show AmTAR1-like immunoreactivity (AmTAR1-IR), suggesting a role of this receptor in modulating sky-compass information and/or higher sensor-motor control. Additionally, intense signals derive from the mushroom bodies, higher-order integration centers for olfactory, visual, gustatory and tactile information. To investigate whether AmTAR1-expressing brain structures are in vicinity to tyramine releasing sites, a specific tyramine antibody was applied. Tyramine-like labeling was observed in AmTAR1-IR positive structures, although it was sometimes weak and we did not always find a direct match of ligand and receptor. Moreover, tyramine-like immunoreactivity was also found in brain regions without AmTAR1-IR (optic lobes, antennal lobes), indicating that other tyramine-specific receptors may be expressed there.

Highly efficient siRNA delivery from core-shell mesoporous silica nanoparticles with multifunctional polymer caps.

A new general route for siRNA delivery is presented combining porous core-shell silica nanocarriers with a modularly designed multifunctional block copolymer. Specifically, the internal storage and release of siRNA from mesoporous silica nanoparticles (MSN) with orthogonal core-shell surface chemistry was investigated as a function of pore-size, pore morphology, surface properties and pH. Very high siRNA loading capacities of up to 380 µg per mg MSN were obtained with charge-matched amino-functionalized mesoporous cores, and release profiles show up to 80% siRNA elution after 24 h. We demonstrate that adsorption and desorption of siRNA is mainly driven by electrostatic interactions, which allow for high loading capacities even in medium-sized mesopores with pore diameters down to 4 nm in a stellate pore morphology. The negatively charged MSN shell enabled the association with a block copolymer containing positively charged artificial amino acids and oleic acid blocks, which acts simultaneously as capping and endosomal release agent. The potential of this multifunctional delivery platform is demonstrated by highly effective cell transfection and siRNA delivery into KB-cells. A luciferase reporter gene knock-down of up to 80-90% was possible using extremely low cell exposures with only 2.5 µg MSN containing 0.5 µg siRNA per 100 µL well.

Mesoporosity--a new dimension for zeolites.

Frameworks of precisely defined pores with diameters matching the size of small molecules endow crystalline zeolites with valuable size- and shape-selectivity. Being important selective adsorbers and separators, zeolites are also indispensable as solid acids in size-selective catalysis. However, despite being extremely beneficial, micropores impose restrictions on the mass transport of reactants, especially when bulky molecules are involved. The prospect to boost the catalytic power of zeolites and to extend their applications into new areas has prompted numerous efforts to synthesize mesoporous zeolitic materials that combine diffusional pathways on two different size scales. Our tutorial review will introduce the reader to this exciting recent development in zeolite science. We will give a general overview of the diverse strategies on how to implement a secondary pore system in zeolites. We will distinguish top-down from bottom-up and template-assisted from 'template-free' procedures. Advantages and limitations of the different methods will also be addressed.

Nanofusion: mesoporous zeolites made easy.

A new path to zeolites: Nanofusion is a template-free, one-step process that gives mesoporous zeolite beta with tunable mesopore diameters in very short reaction times. The hierarchical materials are built from zeolite beta nanocrystals in concentrated gels. The nanocrystals can either be retrieved as individual particles in a colloidal suspension or are directly fused into mesoporous zeolitic materials (see scheme).

One-step synthesis of hierarchical zeolite beta via network formation of uniform nanocrystals.

A hierarchical mesoporous network of zeolite beta with very high micropore as well as mesopore volume was synthesized without the need of a porogen at near 100% yield in the form of easily retrievable micrometer-sized particles. This was achieved by a dense-gel synthesis utilizing steam-assisted conversion (SAC) to induce a burst of nucleation. During the first phase of the synthesis, individual, evenly sized zeolite beta nanoparticles are formed that subsequently condense into a porous network displaying uniform mesopores. The final product consists of hierarchical self-sustaining macroscopic zeolite aggregates assembled from 20 nm crystalline domains of zeolite beta. The small size of the zeolite crystals in the resulting materials gives rise to mesopores with dominant pore sizes of about 13 nm. Large surface areas between 630 and 750 m(2)/g and total pore volumes up to 0.9 mL/g were obtained without sacrificing the microporosity (usually larger than 0.20 mL/g). Crystallization conditions were optimized for different Si/Al ratios between 10 and 33. A complete conversion into hierarchical zeolite beta was achieved in only a few hours at 170-180 °C if the amount of water present during the steam-assisted conversion was adequately adjusted. This dense gel steam conversion process proves to be a highly efficient strategy for fabricating hierarchical zeolite beta networks in a single step.

Colloidal suspensions of functionalized mesoporous silica nanoparticles.

The synthesis and characterization of colloidal mesoporous silica (CMS) functionalized with vinyl-, benzyl-, phenyl-, cyano-, mercapto-, aminopropyl- or dihydroimidazole moieties is reported. Uniform mesoporous particles ranging in size from 40 to 150 nm are generated in a co-condensation process of tetraethylorthosilicate (TEOS) and organotriethoxysilanes (RTES) in alkaline aqueous media containing triethanolamine (TEA) in combination with cetyltrimethylammonium chloride (CTACl) serving as a structure-directing agent. The materials are obtained as colloidal suspensions featuring long-term stability after template removal by ion exchange with an ethanolic solution of ammonium nitrate or HCl. The spherical particles exhibit a wormlike pore system with defined pore sizes and high surface areas. Samples are analyzed by a number of techniques including TEM, SEM, DLS, TGA, Raman, and cross-polarized (29)Si-MAS NMR spectroscopy, as well as nitrogen sorption measurements. We demonstrate that co-condensation and grafting methods result in similar changes in the nitrogen adsorption behavior, indicating a successful internal lining of the pores with functional groups through both procedures.

Water-molecule network and active-site flexibility of apo protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 A apo PTP1B structure has been obtained, showing four highly coordinated water molecules in the active-site pocket of the enzyme; hence, the active site is highly solvated in the apo state. Three of the water molecules are located at positions that approximately correspond to the positions of the phosphate O atoms of the natural substrate phosphotyrosine and form a similar network of hydrogen bonds. The active-site WPD-loop was found to be in the closed conformation, in contrast to previous observations of wild-type PTPs in the apo state, in which the WPD-loop is open. The closed conformation is stabilized by a network of hydrogen bonds. These results provide new insights into and understanding of the active site of PTP1B and form a novel basis for structure-based inhibitor design.

Structure-based design of selective and potent inhibitors of protein-tyrosine phosphatase beta.

Protein-tyrosine phosphatases (PTPs) are considered important therapeutic targets because of their pivotal role as regulators of signal transduction and thus their implication in several human diseases such as diabetes, cancer, and autoimmunity. In particular, PTP1B has been the focus of many academic and industrial laboratories because it was found to be an important negative regulator of insulin and leptin signaling, and hence a potential therapeutic target in diabetes and obesity. As a result, significant progress has been achieved in the design of highly selective and potent PTP1B inhibitors. In contrast, little attention has been given to other potential drug targets within the PTP family. Guided by x-ray crystallography, molecular modeling, and enzyme kinetic analyses with wild type and mutant PTPs, we describe the development of a general, low molecular weight, non-peptide, non-phosphorus PTP inhibitor into an inhibitor that displays more than 100-fold selectivity for PTPbeta over PTP1B. Of note, our structure-based design principles, which are based on extensive bioinformatics analyses of the PTP family, are general in nature. Therefore, we anticipate that this strategy, here applied to PTPbeta, in principle can be used in the design and development of selective inhibitors of many, if not most PTPs.

Radiographic evaluation of osseointegration and loosening of titanium implants in the MCP and PIP joints.

PURPOSE: Good to excellent clinical results have been shown in the initial studies on osseointegrated metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joint implants that consist of a flexible silicone spacer connected to osseointegrated titanium fixtures. A high fracture rate of the silicone spacer, however, has been reported, diminishing the value of this implant system. The purpose of this study was to evaluate the osseointegration of the titanium fixture itself in a large study group and compare patients with rheumatoid arthritis with those with osteoarthrosis as a base for further development of the implant concept. METHODS: Two hundred thirty-nine implants (27 PIP, 212 MCP) in 86 patients were retrospectively evaluated radiographically. Two hundred implants were in patients with rheumatoid arthritis. Radiographic evaluation included measuring the length and width of resorption zones around the titanium fixtures. A scoring system was used that grouped observations in 4 groups, from 0 (little or no change) to 3 (loosening). Status of the silicone spacer was also noted. RESULTS: At follow-up evaluation an average of 41 months (range, 12-103 months) after surgery, complete osseointegration was found in 450 of 478 fixtures (94%). Loosening was found in 10 proximal and 18 distal fixtures. The proximal fixtures showed loosening mainly during the first 3 years after surgery and the distal fixtures also had loosening. In the 31 MCP joint implants that were evaluated for more than 5 years, the osseointegration rate was 97%. CONCLUSIONS: Implant stem fixation with osseointegrated titanium fixtures is a valuable method that works well in rheumatoid arthritis patients. Further development will focus on a more durable constrained joint mechanism.

Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor.

Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.

Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.

Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.

Enzyme kinetic characterization of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.

Structure determination of T cell protein-tyrosine phosphatase.

Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130-132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.