Development of cell-specific RNAi delivery vectors based on poly(β-amino ester)s with therapeutic applications

Els ARNs de interferència han demostrat tenir un potencial terapèutic molt prometedor per tractar malalties causades per desregulació gènica. S’han estudiat i desenvolupat diferents tècniques d’alliberació d’ARNs, tot i així, els dubtes sobre la seva seguretat, especificitat cel·lular i eficiència de transfecció fan necessari el desenvolupament de metodologies d’alliberació alternatives, capaces de conduir de forma específica i segura els àcids nucleics fins a la seva diana terapèutica. Recentment, formulacions polimèriques basades en poly(β-amino ester)s (pBAEs) han sorgit com una opció per l’alliberació d’àcids nucleics degut a la seva baixa toxicitat, alta biocompatibilitat i formulació química simple. Concretament, en aquest treball, s’ha desenvolupat una nova família de pBAEs incorporant-los-hi en els seus extrems oligopèptids formats per aminoàcids catiònics i aniònics, capaços de condensar ARNs tot obtenint partícules amb mides manomètriques. A més, s’ha demostrat que variant la hidrofobicitat de la seva cadena central, s’obtenen formulacions polimèriques amb diferents propietats biofísiques. Tenint en compte les característiques prèviament descrites, s’han dissenyat sistemes d’alliberació d’ARNs amb una major capacitat d’emmagatzematge, estabilitat i eficàcia de transfecció. A més, s’ha observat que jugant amb la composició oligopeptidica dels pBAEs és possible controlar la seva especificitat cel·lular, tant a nivell in vitro com in vivo. Conseqüentment, aquets nous sistemes d’alliberació mostren un elevat potencial pel tractament de línies cel·lulars difícils o delicades de transfectar. Per exemple, la combinació de pBAEs modificats amb arginina i àcid aspàrtic formen poliplexes capaços d’alliberar eficientment anti-OCT3/4 siRNA, anti-NANOG siRNA i RUNX2 plasmid en Dental Pulp Pluripotent Stem Cells, millorant la seva diferenciació osteogènica. Per altra banda, a nivell in vivo, s’ha observat que la combinació de pBAEs modificats amb lisina i histidina forma poliplexes capaços d’alliberar preferencialment àcids nucleics sobre cèl·lules endotelials de la vasculatura. Finalment, s’han optimitzat les nanopartícules amb la finalitat d’alliberar ARNs mitjançant endocitosis mediada per receptor sobre cèl·lules malaltes evitant efectes no desitjats sobre les cèl·lules sanes del mateix teixit. Mitjançant un assaig in vivo utilitzant un model d’ateroesclerosis en ratolí, s’ha demostrat que el sistema d’alliberament dissenyat és capaç de regular l’expressió gènica en cèl·lules endotelials inflamades. En conclusió, aquesta tesi demostra que un disseny acurat en la formació dels poliplexes d’ ARNs permet la fabricació de sistemes d’alliberació estables, específics i eficients, donant com a resultat, vectors d’alliberació fàcilment adaptables en funció de la seva aplicació terapèutica.Los ARNs de interferencia han demostrado tener un potencial terapéutico muy prometedor para el tratamiento de enfermedades debidas a la desregulación génica. Se han estudiado y desarrollado distintas técnicas de liberación de ARNs, sin embargo, las dudas sobre su seguridad, especificidad celular y eficiencia de transfección hacen necesario el desarrollo de metodologías de liberación alternativas, capaces de conducir de forma selectiva y segura los ARNs de interferencia hasta su diana. Recientemente, formulaciones poliméricas basadas en poly(β-amino ester)s (pBAEs) han surgido como una prometedora alternativa para la liberación de ácidos nucleicos, debido a su baja toxicidad, alta biocompatibilidad y simple formulación química. Concretamente, en este trabajo, se ha sintetizado una nueva familia de pBAEs que incorpora diferentes oligopéptidos catiónicos y aniónicos, capaces de condensar ARNs en partículas con tamaños nanométricos. Además, se ha demostrado que variando la hidrofobicidad de su estructura, se obtienen formulaciones poliméricas con distintas propiedades biofísicas. Considerando todos estos factores, se han diseñado sistemas de liberación de ARNs con una mayor capacidad de almacenamiento, estabilidad y eficacia de transfección. Además, se ha observado que controlado la composición oligopeptidica de los pBAEs es posible formular poliplejos con una elevada especificad celular, tanto a nivel in vitro como in vivo. Consecuentemente, estos nuevos sistemas de liberación muestran un elevado potencial para el tratamiento de líneas celulares difíciles o delicadas de transfectar. Por ejemplo, la combinación de pBAEs modificados con arginina y ácido aspártico forman poliplejos capaces de liberar eficientemente y simultáneamente anti-OCT3 / 4 siRNA, anti-NANOG siRNA y RUNX-2 plásmido sobre Dental Pulp Pluripotent Stem Cells, mejorando su diferenciación osteogénica. Por otra parte, a nivel in vivo, se ha observado que la combinación de pBAEs modificados con lisina e histidina forma poliplejos capaces de liberar preferencialmente ácidos nucleicos sobre células endoteliales de la vasculatura. Finalmente, los poliplejos se han optimizado con el fin de liberar ARNs mediante endocitosis mediada por receptor sobre células enfermas, evitando efectos no deseados sobre las células sanas del mismo tejido. Mediante un ensayo in vivo utilizando un modelo de ateroesclerosis en ratón, se ha demostrado que el sistema de liberación diseñado es capaz de regular la expresión génica sobre células endoteliales inflamadas. En conclusión, esta tesis demuestra que un cuidadoso diseño en la formulación de los poliplejos de ARN permite la fabricación de sistemas de liberación estables, específicos y eficientes, dando como resultado, vectores de liberación adaptables en función de la su aplicación terapéutica.RNAi technology has gained rapid promise for its potential therapeutic application for diseases caused by abnormal gene overexpression. Although RNAi delivery strategies have been widely studied and developed, concerns about their safety, cell-specificity and efficiency delivering prompts the development of alternative delivery methodologies capable of efficiently drive RNAi-based nucleic acids to the target cells in a safe manner. Recently, newly developed poly(β-amino ester)s (pBAEs) have emerged as an interesting choice for nucleic acid delivery due to their low toxicity, high biocompatibility, and simple chemical formulation. Here, we present an extended family of pBAEs that incorporate terminal oligopeptide moieties, containing both positive and negative amino acids, able to condense RNAi-based nucleic acids into discrete nanoparticles. Furthermore, we demonstrated that pBAE backbone hydrophobicity has a deep effect in the resulting polyplexes formulations. With this new approach, we have obtained delivery formulations with increased RNAi packaging capacity, stability, and transfection efficiency. In addition, in vitro and in vivo results have demonstrated that careful control of the pBAE oligopeptide composition is a powerful strategy to efficiently deliver nucleic acids in a cell-type dependent manner. These delivery vectors have specially shown great promise for difficult-to-transfect cells. For instance, pBAE formulations using a combination of arginine and aspartic acid oligopeptides were able to efficiently and simultaneously deliver anti-OCT3/4 siRNA, anti-NANOG siRNA, and RUNX2 plasmid to Dental Pulp Pluripotent Stem Cells in order to promote their osteogenic differentiation. At in vivo level, it has been found that a combination of lysine- and histidine- modified pBAEs was able to preferentially deliver siRNA to endothelial cells from the vasculature with low off-target effects. Finally, polyplexes have been further optimized to selectively deliver RNAi-based nucleic acids via receptor-mediated endocytosis in order to selectively target diseased cells, while avoiding healthy cells populations from the same tissue. In vivo results demonstrated that the targeted delivery system proposed here was able to efficiently regulate gene expression in inflamed endothelial cells from atherosclerotic mice model, obtaining a promising therapeutic effect. In conclusion, this thesis demonstrates that careful control of the composition of pBAE-nucleic acid formulations allows the fabrication of stable, specific and efficient delivery systems, resulting in promising delivery vectors that can be easily adapted for specific therapeutic applications

Effect of the liquid layer on the impact behaviour of particles

During a spray granulation process the moisture loading in fluidized beds has a great influence on\ud the inter-particle collision properties and hence on the flow behaviour. To study the influence of the\ud liquid layer as well as granule impact velocity on the impact behaviour free-fall experiments were\ud performed. During these experiments the g-Al2O3 granules were dropped from a predefined height\ud onto a liquid layer on the flat steel wall and the velocity-time curves were obtained using highspeed\ud video recording. The height of the liquid layer was varied from 50 mm to 1 mm. Moreover,\ud the tests were performed at different velocities and viscosities of liquid layer in the range of 1-300\ud mPa∙s. Both distilled water and water solutions of hydroxypropyl methylcellulose with different\ud concentrations (3, 6, 10 mass-%) were used.\ud The obtained restitution coefficients were compared with the experiments performed without liquid\ud film on the surface. For a granule impacted on a liquid film on the wall, the increase of liquid\ud viscosity decreases the restitution coefficient and thickness of liquid layer at which the granule\ud sticks. In the examined velocity range, with decreasing impact velocity the restitution coefficient\ud greatly decreases. To explain the obtained effects the force and energy balances for a particle\ud impacted on a liquid layer on the wall were derived. Both contributions to energy absorption\ud (granule-liquid layer and granule-wall contacts) have been taken into consideratio

Editorial: Biofuels and Bioproducts From Anaerobic Processes: Anaerobic Membrane Bioreactors (AnMBRs)

New biodegradable waste treatment configurations and technologies have arisen to support the transition of treatment plants toward resource recovery facilities. The interest in Anaerobic Membrane Biological Reactor (AnMBR) technology is increasing due to the advantages related to combine anaerobic digestion with membrane filtration. Thanks to the complete retention of anaerobic microorganisms, AnMBRs have the capacity to efficiently recover most of the energy potential in biodegradable waste streams in the form of biogas and produce high-quality effluents with low biomass production

Wear and corrosion of metal-matrix (stainless steel or NiTi)-TiC coatings

AbstractDifferent spraying technologies (plasma and High Velocity Oxi-Fuel) have been use to obtain TiC - stainless steel or NiTi matrix coatings. The starting feedstock powders have been obtained by SHS technology. After crushing and sieving, the fraction of particles between 20 and 63μm, have been selected for thermal spray. The obtained coatings have been characterized by XRD and SEM-EDS to observe the surface and cross section. The coatings adhesion, wear (ball-on-disk and rubber wheel tests) and electrochemical corrosion test have been carried out.Results show that the plasma sprayed coatings with NiTi have better adhesion than the stainless steel matrix coatings. However, the opposite happens for HVOF coatings. NiTi matrix coatings exhibit higher wear resistance both for plasma and HVOF spraying processes

Using of spouted bed spray granulation process for fabricating of metal/ceramic-polymer composites

Naturally occurring structural materials are composites with very high filling degrees on hard constituent. In recent years structure and properties of biological materials have been studied in detail. But it has not been possible to reconstruct this structural design. In this contribution we present a process for the fabrication of very highly filled composite materials by using of the spouted bed spray granulation process. Spouted bed granulation offers many advantages for the design of composite materials. In this investigation fine particles (dP =10-50 µm) could be uniformly spouted, and optimal properties for further processing to bulk materials can be obtained by means of granulation. For this a hybrid spouted bed with horizontal gas inlets was designed, which has a small prismatic process chamber with adjustable inlets and a high conical-cylindrical relaxation zone. The thickness of the polymer layer is adjusted by a two-fluid nozzle. The adjustment of the thickness of polymer layer is very important to fabricate composites of adjustable filling degrees. After granulation the particles are assembled to a composite material by means of warm pressing. Additionally for achievement of very high packing densities bimodal particle size distributions are used. For this purpose, particles in nm-range are firstly suspended in polymer solution and the suspension is sprayed in the spouted bed on coarser particles. Granulated particles are assembled to composites and mechanical properties of these were analysed by 4-point bending-tests. We gratefully acknowledge financial support from the German Research Foundation (DFG) via the collaborative research center SFB986. Please click Additional Files below to see the full abstract

Hierarchical Coarse-Grained Strategy for Macromolecular Self-Assembly: Application to Hepatitis B Virus-Like Particles

Macromolecular self-assembly is at the basis of many phenomena in material and life sciences that find diverse applications in technology. One example is the formation of virus-like particles (VLPs) that act as stable empty capsids used for drug delivery or vaccine fabrication. Similarly to the capsid of a virus, VLPs are protein assemblies, but their structural formation, stability, and properties are not fully understood, especially as a function of the protein modifications. In this work, we present a data-driven modeling approach for capturing macromolecular self-assembly on scales beyond traditional molecular dynamics (MD), while preserving the chemical specificity. Each macromolecule is abstracted as an anisotropic object and high-dimensional models are formulated to describe interactions between molecules and with the solvent. For this, data-driven protein–protein interaction potentials are derived using a Kriging-based strategy, built on high-throughput MD simulations. Semi-automatic supervised learning is employed in a high performance computing environment and the resulting specialized force-fields enable a significant speed-up to the micrometer and millisecond scale, while maintaining high intermolecular detail. The reported generic framework is applied for the first time to capture the formation of hepatitis B VLPs from the smallest building unit, i.e., the dimer of the core protein HBcAg. Assembly pathways and kinetics are analyzed and compared to the available experimental observations. We demonstrate that VLP self-assembly phenomena and dependencies are now possible to be simulated. The method developed can be used for the parameterization of other macromolecules, enabling a molecular understanding of processes impossible to be attained with other theoretical models

Simultaneous ammonium and phosphate recovery and stabilization from urban sewage sludge anaerobic digestates using reactive sorbents

The use of low-cost inorganic sorbents as a new sustainable strategy to enhance the valorization of nutrients (N-P-K), from the urban water cycle (e.g., side streams from sewage sludge anaerobic digestion), in agriculture applications is presented. The simultaneous recovery and stabilization of ammonium and phosphate by using a mixture of two reactive sorbents (Na and K zeolites and magnesium oxide) was evaluated. The nutrients stabilization process, favoured at alkaline pH values, is carried out by a) the precipitation of phosphate ions with magnesium and/or ammonium ions and b) the sorption of ammonium by Na- and K-zeolites. MgO(s) promoted the stabilization of phosphate as bobierrite (Mg3(PO4)2(s)) or struvite (MgNH4PO4(s)) depending on the applied dose. Doses with the stoichiometric molar ratio of Mg/P promote the formation of bobierrite, while molar ratios higher than 3 favour the formation of struvite. Na zeolites (NaP1-NA, NaP1-IQE) demonstrated efficiency on ammonium stabilization between 60 ± 2 (for 15 gZ/L) to 90 ± 3% (for 50 gZ/L). The ammonium recovery efficiency is limited by the zeolite sorption capacity. If the target of the fertilizing criteria should include K, then the use of a K-zeolite (e.g., 5AH-IQE) provides a good solution. The optimum pH for the precipitation of struvite and bobierrite is 9.5 and the optimum pH for ammonium removal is between 4 and 8.5. N is present in higher concentrations (up 0.7–1 g NH4+/L) when pH is ranged between 8.2 and 8.6. The ammonium recovery ratios were better than those previously reported using only magnesium oxide or even a more expensive reagent as newberrite (MgHPO4(s)). The recovery mechanisms described generate low-solubility stabilized nutrients forms that potentially can be applied as slow-release fertilizers in agriculture. Thus, the use in agriculture of blends of digested sludge with low-solubility stabilized nutrients forms will improve soils quality properties in terms of organic matter and nutrients availability.Peer ReviewedPostprint (author's final draft

Volatile fatty acid production from mesophilic acidogenic fermentation of organic fraction of municipal solid waste and food waste under acidic and alkaline pH

This study is focused on the effects of pH on the production of volatile fatty acids (VFAs) and their distribution through the acidogenic fermentation of source-sorted organic fraction of municipal solid waste (OFMSW) from a mechanical-biological treatment (MBT) plant, and food waste (FW) from a university canteen. In semi-continuous lab-scale digesters using OFMSW at a hydraulic retention time (HRT) of 3.5 days under acidic conditions (pH 6.0), the VFA concentration in the effluent increased to 9.8-11.5 g L−1 (VS content of the feedstock between 4.2 and 5.2% w/w), while its individual VFA profiling was similar to the influent which was already prefermented (namely, C2 35-41%, C3 18-22%, C4 17-21%, and C5 9-12%). When working with the same conditions but using FW as feedstock, an effluent with a VFA concentration up to 11.5 g VFA L−1 (FW with a VS content of 5.5% w/w) and a stable distribution of C2 and C4 acids (up to 60.3% and 12.9%, respectively) but with very low quantities of C3 and C5 acids (lower than 1.8 and 2.7%, respectively) was obtained. Anaerobic batch tests using FW revealed that alkaline pH near 9 could lead to higher VFA production with high acetic acid content when compared to pH 6. In the semi-continuous fermenters working at alkaline conditions (pH 9.5-10) using OFMSW and FW, an enhanced solubilization of organic matter was registered with respect to the fermenters working under acidic conditions. This fact was not reflected in a higher VFA production when using OFMSW as feedstock, probably due to free ammonia inhibition, since OFMSW was mixed in the MBT plant with supernatant from anaerobic digestion of this biowaste. However, when using FW, alkaline conditions lead to an enhanced VFA production with respect to the reactor working under acidic conditions, being acetic acid the predominant product, which represented up to 91% of the VFA spectrum obtained

Development of Bioglass/PEEK Composite Coating by Cold Gas Spray for Orthopedic Implants

Cold gas spray (CGS) technology has allowed the development of biofunctional coatings composed of 45S5 and polyetheretherketone (PEEK). The combination of a bioactive glass material embedded in a polymeric matrix makes this composite an interesting material for orthopedic applications since this composite meets the biomechanical and biological requirements of an implant. In the present study, blends of bioactive glass 45S5 and PEEK powder with different granulometry and 45S5/PEEK ratio have been prepared. These mixtures of powders have been deposited onto PEEK substrates by CGS with the goal of incorporating a bioactive additive to the biocompatible polymer, which can improve the bone-implant interaction of PEEK. The deposition efficiency (DE) of the coatings has been evaluated, and from the results obtained, it was possible to conclude that DE is significantly affected by the granulometry and by the 45S5/PEEK ratio of the blends. By scanning electron microscopy (SEM) inspection, it was observed that the use of blends with high 45S5/PEEK ratio lead to the deposition of coatings with high content of 45S5. Finally, the friction behavior of the coatings was analyzed performing ball-on-disk tests and these experiments showed that the presence of glass particles has a beneficial role in the wear resistance