RESUMEN
Polystyrene (PS) is an important model polymer for the investigation of effects of microplastic (MP) and nanoplastic (NP) particles on living systems. Aqueous dispersions of PS MP or NP contain residual monomers of styrene. In consequence, it is not clear if the effects observed in standard (cyto)toxicity studies are evoked by the polymer (MP/NP) particle or by residual monomers. We addressed that question by comparing standard PS model particle dispersions with in-house synthesized PS particle dispersions. We proposed a rapid purification method of PS particle dispersions by dialysis against mixed solvents and developed a simple method of UV-vis spectrometry to detect residual styrene in the dispersions. We found that standard PS model particle dispersions, which contain residual monomers, exerted a low but significant cytotoxicity on mammalian cells, while the in-house synthesized PS, after rigorous purification to reduce the styrene content, did not. However, the PS particles per se but not the residual styrene in both PS particle dispersions resulted in immobilization of Daphnia. Only by using freshly monomer-depleted particles, will it be possible in the future to assess the (cyto)toxicities of PS particles, avoiding an otherwise not controllable bias effect of the monomer.
Asunto(s)
Microplásticos , Poliestirenos , Animales , Poliestirenos/toxicidad , Microplásticos/toxicidad , Plásticos , Polímeros , Solventes , MamíferosRESUMEN
BACKGROUND: One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. RESULTS: Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. CONCLUSIONS: Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098).
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Magnetosomas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomineralización/genética , Óxido Ferrosoférrico/análisis , Óxido Ferrosoférrico/metabolismo , Magnetosomas/genética , Magnetosomas/metabolismo , Magnetospirillum , Factor sigma/genética , TranscriptomaRESUMEN
Microplastic particles (MP) and nanoplastic particles (NP) as persistent anthropogenic pollutants may impact environmental and human health. A relevant potential source of primary MP and NP is water-based dispersion paint which are commonly used in any household. Given the worldwide high application volume of dispersion paint and their diverse material composition MP and NP may enter the environment with unforeseeable consequences. In order to understand the relevance of these MP and NP from paint dispersion we investigated the components of two representative wall paints and analyzed their composition in detail. The different paint components were then investigated for their impact on the model organism Daphnia magna and on a murine cell line. Plastic NP, dissolved polymers, titanium dioxide NPs, and calcium carbonate MPs demonstrated adverse effects in both biological test systems, indicating detrimental consequences of several typical components of wall paints upon release into the environment. The outcome of this study may form the basis for the evaluation of impact on other organisms, environmental transport and impact, other related technical materials and for the development of strategies for the prevention of potential detrimental effects on organisms.
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Microplásticos , Contaminantes Químicos del Agua , Animales , Daphnia , Humanos , Ratones , Pintura/toxicidad , Plásticos/toxicidad , Polímeros , Contaminantes Químicos del Agua/análisisRESUMEN
The earthworm Eisenia fetida is a commonly used model organism for unspecific soil feeders in ecotoxicological studies. Its intestinal cells are the first to encounter possible pollutants co-ingested by the earthworm, which makes them prime candidates for studies of toxic effects of environmental pollutants on the cellular as compared to the organismic level. In this context, the aim of this study was to demonstrate the suitability of preparations of primary intestinal E. fetida cells for in vitro ecotoxicological studies. For this purpose, a suitable isolation and cultivation protocol was established. Cells were isolated directly from the intestine, maintaining >85% viability during subsequent cultivations (up to 144 h). Exposure to established pollutants and soil elutriates comprising silver nanoparticles and metal ions (Cu2+, Cd2+) induced a significant decrease in the metabolic activity of the cells. In case of microplastic particles (MP particles), namely 0.2, 0.5, 2.0, and 3.0 µm diameter polystyrene (PS) beads as well as 0.5 and 2.0 µm diameter polylactic acid (PLA) beads, no active uptake was observed. Slight positive as well as negative dose and size dependent effects on the metabolism were seen, which to some extent might correlate with effects on the organismic level.
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Nanopartículas del Metal , Oligoquetos , Contaminantes del Suelo , Animales , Intestinos/química , Nanopartículas del Metal/toxicidad , Plásticos/metabolismo , Plásticos/farmacología , Plata/metabolismo , Suelo , Contaminantes del Suelo/análisisRESUMEN
Biogas has the potential to contribute to some of the most urgent issues of the energy transition, including mobility, energy storage, and grid stability. Flexibilization has been discussed as a means to improve the economics of biogas production, ideally restricting the production of electricity to times of strong need. Here the possibility of demand-driven, flexible biogas production is investigated, which saves substrates and storage capacity, while still enabling control over the production of electricity. Effects of different flexible feeding regimes were tested in a continuously operated 200 L reactor. After a period of 300 days under steady conditions (6.4 kg feed m-3d-1), varying flexible feeding patterns were applied over the next 700 days. Biogas production, volatile organic acid concentrations, and microbial dynamics were documented. Reduction of feeding resulted in reducing the gas production by up to 80% within a day. By increasing the feed, gas production could rapidly be reinitiated at similar levels as before even after fasting periods of up to 22 days. CH4-contents of the produced biogas were nearly constant over the investigation period. As a response to the flexible feeding, a reorganization of the microbial community was observed, which came to an end after 800 days and then was no longer affected by further changes in the feeding patterns or the substrate composition. Dominating archaea were of the order Methanosarcinales. During the experiment, representatives from the class Methanosaetaceae replaced representatives from the class Methanosarcinaceae.
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Archaea/metabolismo , Gases/metabolismo , Microbiota , Anaerobiosis , Archaea/clasificación , Archaea/genética , Archaea/aislamiento & purificación , Reactores Biológicos/microbiología , Digestión , Metano/análisis , Metano/metabolismo , Aguas del AlcantarilladoRESUMEN
Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current "gold standard", namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.
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Linfocitos B/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Linfocitos B/citología , Células Cultivadas , Humanos , Metacrilatos/química , Nylons/química , TransgenesRESUMEN
A major challenge in tissue engineering and artificial scaffolding is to combine easily tunable scaffolds biomimicking the extracellular matrix of native organs with delivery-controlled cell culturing to create fully cellularized, large artificial 3D scaffolds. Aiming at bioartificial liver construction, we present our research using galactose-functionalized, ultraporous polylactide 3D nanofiber sponges fabricated out of electrospun fibers. Sponge biomodification by blend galactosylation and in-solution coating is performed, respectively, using a polylactide-galactose carrier-copolymer that promotes cell delivery and features a pronounced autofluorescence. It allows us to verify the galactosylation success, evaluate its quality, and record dye-free, high-resolution images of the sponge network using confocal laser scanning microscopy. The galactose carrier and its impact on scaffold cellularization is validated in benchmark to several reference systems. Verification of the human hepatic cell asialoglycoprotein receptor presence and galactose interaction in culture is performed by Cu2+ receptor-blocking experiments. The culture results are extensively investigated in and ex situ to trace and quantify the cell culture progress, cell activity, and viability at different culture stages. Bioreactor cultivation of sponges reveals that the galactose carrier does not only facilitate cell adhesion but also enhances cellular distribution throughout the scaffold. The promising 3D culture results allow us to move forward to create mature in vitro liver model research systems. The elaboration into ex vivo testing platforms could help judging native cell material interactions with drugs or therapeutics, without the need of direct human or animal testing.
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Hígado Artificial , Ingeniería de Tejidos , Animales , Matriz Extracelular , Humanos , Perfusión , Polímeros , Andamios del TejidoRESUMEN
BACKGROUND: Magnetosomes produced by magnetotactic bacteria represent magnetic nanoparticles with unprecedented characteristics. However, their use in many biotechnological applications has so far been hampered by their challenging bioproduction at larger scales. RESULTS: Here, we developed an oxystat batch fermentation regime for microoxic cultivation of Magnetospirillum gryphiswaldense in a 3 L bioreactor. An automated cascade regulation enabled highly reproducible growth over a wide range of precisely controlled oxygen concentrations (1-95% of air saturation). In addition, consumption of lactate as the carbon source and nitrate as alternative electron acceptor were monitored during cultivation. While nitrate became growth limiting during anaerobic growth, lactate was the growth limiting factor during microoxic cultivation. Analysis of microoxic magnetosome biomineralization by cellular iron content, magnetic response, transmission electron microscopy and small-angle X-ray scattering revealed magnetosomal magnetite crystals were highly uniform in size and shape. CONCLUSION: The fermentation regime established in this study facilitates stable oxygen control during culturing of Magnetospirillum gryphiswaldense. Further scale-up seems feasible by combining the stable oxygen control with feeding strategies employed in previous studies. Results of this study will facilitate the highly reproducible laboratory-scale bioproduction of magnetosomes for a diverse range of future applications in the fields of biotechnology and biomedicine.
Asunto(s)
Automatización de Laboratorios , Fermentación , Magnetosomas/metabolismo , Magnetospirillum/crecimiento & desarrollo , Magnetospirillum/metabolismo , Oxígeno/metabolismo , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Biotecnología , Carbono/metabolismo , Óxido Ferrosoférrico/metabolismoRESUMEN
SEAP (secreted embryonic alkaline phosphatase) has been suggested as versatile reporter protein inter alia for cell ligand interaction. Generic photometric assay formats for this enzyme are currently lacking. Using the interaction of recombinant hCD40 ligand with HEK-Blue sensor cells expressing the CD40 receptor as example, we show that such an assay can be developed based on BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride) as substrate. Supplementation of the reaction buffer with a micelle-forming detergent (TWEEN 20) stabilizes the water-insoluble reactions products thereby allowing reproducible photometric quantification of the colloidal dispersion. After optimizing the assay in terms of incubation time, cell number and environmental conditions, a cellular response to stimulation was already visible for 0.25â¯ngâ¯mL-1 of rhCD40L. Moreover, the sensitivity of the assay was significantly better than reported previously for alternative assays used in combination with the commercially available reporter cells. The use of BCIP/NBT as substrate therefore provides a robust and sensitive method to monitor SEAP activity in solution, which could conceivably be extended to other cell-based and biological assays using SEAP as reporter protein.
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Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/metabolismo , Indoles/química , Nitroazul de Tetrazolio/química , Técnicas Biosensibles , Ligando de CD40/metabolismo , Línea Celular , Coloides/química , Humanos , Indicadores y Reactivos/química , Ligandos , Límite de Detección , Proteína Cofactora de Membrana/química , FotometríaRESUMEN
A number of evolving medical therapies call for the controlled expansion of primary human T lymphocytes. After encapsulation in sodium cellulose sulfate-poly(diallyldimethyl) ammonium chloride polyelectrolyte capsules, T lymphocytes can be expanded without persisting activation. Here, the challenge of scaling up this process is addressed. Encapsulated T lymphocytes were cultured in spinner flasks as well as in several types of the bioreactor, including fixed and fluidized beds, a waved cell bag, and a standard stirred tank reactor (STR; 1-L scale). Two proprietary T lymphocyte culture media as well as a standard RPMI-based medium were used. As before, encapsulation coincided with the presence of only a low fraction of activated T lymphocytes (peripheral blood T cells) in the total population. Unexpectedly, growth rates were lower in well-mixed reactors than those in cultivations under static conditions, that is, in T-flasks. Switching the STR to low oxygen conditions (40% air saturation) improved the growth rates to the level of the static cultures and thus forms the potential basis for efficient scale-up of T lymphocyte expansion.
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Reactores Biológicos , Técnicas de Cultivo de Célula , Proliferación Celular , Células Inmovilizadas/metabolismo , Linfocitos T/metabolismo , Células Inmovilizadas/citología , Medios de Cultivo/química , Humanos , Linfocitos T/citologíaRESUMEN
Ultraporous, degradable sponges made of either polylactide or of blends of polylactide/poly(ε-caprolactone) are prepared by freeze-drying of dispersions of short electrospun fibers and subsequent thermal annealing. The sponges feature ultrahigh porosity (99.6%), a hierarchical cellular structure, and high reversible compressibility with fast recovery from deformation in the dry as well as in the wet state. The sponge properties depend on the fiber dispersion concentration and the annealing temperature. Sponge characteristics like fiber density (2.5-20 mg/cm3), size, shape, crystallinity, mechanical strength, wetability, and structural integrity are user adjustable. Cell culture experiments were successfully performed with Jurkat cells with Confocal Laser Scanning Microscopy and MTT staining showing rapid cell proliferation. Live/Dead staining demonstrated high viability of the seeded cells. The sponge characteristics and modifications investigated and presented here reveal that these sponges are highly promising for tissue engineering applications.
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Hidrogeles/síntesis química , Poliésteres/química , Andamios del Tejido/química , Proliferación Celular/efectos de los fármacos , Fuerza Compresiva , Humanos , Hidrogeles/farmacología , Células Jurkat , Porosidad , Ingeniería de Tejidos/métodos , HumectabilidadRESUMEN
Smart polymers are a valuable platform to protect and control the activity of biological agents over a wide range of conditions, such as low pH, by proper encapsulation. Such conditions are present in olive oil mill wastewater with phenol as one of the most problematic constituents. We show that elastic and pH-responsive diblock copolymer fibers are a suitable carrier for Corynebacterium glutamicum, i.e., bacteria which are known for their ability to degrade phenol. Free C. glutamicum does not survive low pH conditions and fails to degrade phenol at low pH conditions. Our tea-bag like biohybrid system, where the pH-responsive diblock copolymer acts as a protecting outer shell for the embedded bacteria, allows phenol degradation even at low pH. Utilizing a two-step encapsulation process, planktonic cells were first encapsulated in poly(vinyl alcohol) to protect the bacteria against the organic solvents used in the second step employing coaxial electrospinning.
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Biotecnología/métodos , Corynebacterium glutamicum/metabolismo , Fenol/metabolismo , Alcohol Polivinílico/química , Aguas Residuales/química , Purificación del Agua/métodos , BiotransformaciónRESUMEN
Polycations are popular agents for nonviral delivery of DNA to mammalian cells. Adding hydrophobic, biodegradable, or cell-penetrating functions could help to improve their performance, which at present is below that of viral agents. A crucial first step in gene delivery is the complexation of the DNA. The characteristics of these "polyplexes" presumably influence or even determine the subsequent steps of membrane passage, intracellular traveling/DNA release, and nuclear uptake. Herein, polyplexes formed with linear poly(ethylenimine) (l-PEI) are compared to complexes generated with functionalized diblock copolymers. While l-PEI interacts only electrostatically with the DNA, interaction in the case of the diblock polymers may be mixed-mode. In certain cases, transfection efficiency improved when the polyplexes were formed in hypertonic solution. Moreover, whereas conventional PEI-based polyplexes enter the cells via endocytosis, at least one of the diblock agents seemed to promote entry via transient destabilization of the plasma membrane.
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ADN/química , Plásmidos/química , Polietileneimina/química , Animales , Materiales Biocompatibles/química , Células CHO , Línea Celular , Cricetulus , Endocitosis/efectos de los fármacos , Técnicas de Transferencia de Gen , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Tamaño de la Partícula , Poliaminas/química , Polielectrolitos , Electricidad Estática , TransfecciónRESUMEN
The change in thermoresponsive behavior from a single phase transition of upper critical solution temperature (UCST)-type of an acrylamide-acrylonitrile copolymer (AAm-co-AN) to a double responsive behavior (LCST-UCST-type (LCST, lower critical solution temperature)) in water by the introduction of a poly(ethylene glycol) (PEG) block is highlighted in the present work. The polymer is synthesized in a simple way by free-radical polymerization of acrylamide and acrylonitrile using a poly(ethylene glycol) (PEG) macro-azoinitiator. The dual thermoresponsive behavior was observed in a wide range of concentrations repeatable for many cycles with very small hysteresis depending upon the ratio of AAm, AN and PEG. Static light scattering (SLS) and dynamic light scattering (DLS) together with turbidity photometry and transmission electron microscopy confirmed a unique phase transition behavior due to the temperature dependent change in the morphology from micelles to agglomerates. The low cytotoxicity and two-in-one thermoresponsive behavior makes the polymer promising for biomedical applications in the future.
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Resinas Acrílicas/química , Polietilenglicoles/química , Temperatura , Resinas Acrílicas/síntesis química , Estructura Molecular , Tamaño de la Partícula , Polietilenglicoles/síntesis química , Soluciones , Propiedades de SuperficieRESUMEN
Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 µm, 3.6 × 108 particles/mL; 6 µm, 2.1 × 105 particles/mL) and fragments (<30 µm, 5 × 103 particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 µm, while T. pyriformis ingests particles up to 10 µm. In A. proteus, small MP (PS0.5µm and PS6µm) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS<30µm) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS<30µm), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.
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Cadena Alimentaria , Agua Dulce , Microplásticos , Poliestirenos , Contaminantes Químicos del Agua , Contaminantes Químicos del Agua/metabolismo , Monitoreo del Ambiente , Tetrahymena pyriformis/metabolismo , Amoeba/metabolismo , Paramecium caudatum/metabolismo , Tamaño de la PartículaRESUMEN
Due to global pollution derived from plastic waste, the research on microplastics is of increasing public interest. Until now, most studies addressing the effect of microplastic particles on vertebrate cells have primarily utilized polystyrene particles (PS). Other studies on polymer microparticles made, e.g., of polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), or poly (ethylene terephthalate) (PET), cannot easily be directly compared to these PS studies, since the used microparticles differ widely in size and surface features. Here, effects caused by pristine microparticles of a narrow size range between 1 - 4 µm from selected conventional polymers including PS, PE, and PVC, were compared to those of particles made of polymers derived from biological sources like polylactic acid (PLA), and cellulose acetate (CA). The microparticles were used to investigate cellular uptake and assess cytotoxic effects on murine macrophages and epithelial cells. Despite differences in the particles' properties (e.g. ζ-potential and surface morphology), macrophages were able to ingest all tested particles, whereas epithelial cells ingested only the PS-based particles, which had a strong negative ζ-potential. Most importantly, none of the used model polymer particles exhibited significant short-time cytotoxicity, although the general effect of environmentally relevant microplastic particles on organisms requires further investigation.
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Polímeros , Contaminantes Químicos del Agua , Animales , Ratones , Microplásticos , Plásticos , Poliestirenos , Polietileno/análisis , Contaminantes Químicos del Agua/análisis , Monitoreo del AmbienteRESUMEN
Research on the plastic contamination of organic fertilizer (compost) has largely concentrated on particles and fragments > 1 mm. Small, submillimeter microplastic particles may be more hazardous to the environment. However, research on their presence in composts has been impeded by the difficulty to univocally identify small plastic particles in such complex matrices. Here a method is proposed for the analysis of particles between 0.01 and 1.0 mm according to number, size, and polymer type in compost. As a first demonstration of its potential, the method is used to determine large and small microplastic in composts from eight municipal compost producing plants: three simple biowaste composters, four plants processing greenery and cuttings and one two-stage biowaste digester-composter. While polyethylene, PE, tends to dominate among fragments > 1 mm, the microplastic fraction contained more polypropylene, PP. Whereas the contamination with PE/PP microplastic was similar over the investigated composts, only composts prepared from biowaste contained microplastic with a signature of biodegradable plastic, namely poly(butylene adipate co-terephthalate), PBAT. Moreover, in these composts PBAT microplastic tended to form the largest fraction. When the bulk of residual PBAT in the composts was analyzed by chloroform extraction, an inverse correlation between the number of particles > 0.01 mm and the total extracted amount was seen, arguing for breakdown into smaller particles, but not necessarily a mass reduction. PBAT oligomers and monomers as possible substrates for subsequent biodegradation were not found. Remaining microplastic will enter the environment with the composts, where its subsequent degradability depends on the local conditions and is to date largely uninvestigated.
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Compostaje , Plásticos , Plásticos/análisis , Microplásticos , Polímeros , PolipropilenosRESUMEN
Monodisperse, magnetic nanoparticles as vectors for gene delivery were successfully synthesized via the grafting-from approach. First, oleic acid stabilized maghemite nanoparticles (γ-Fe2O3) were encapsulated with silica utilizing a reverse microemulsion process with simultaneous functionalization with initiating sites for atom transfer radical polymerization (ATRP). Polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA) from the core-shell nanoparticles led to core-shell-corona hybrid nanoparticles (γ-Fe2O3@silica@PDMAEMA) with an average grafting density of 91 polymer chains of DP(n) = 540 (PDMAEMA540) per particle. The permanent attachment of the arms was verified by field-flow fractionation. The dual-responsive behavior (pH and temperature) was confirmed by dynamic light scattering (DLS) and turbidity measurements. The interaction of the hybrid nanoparticles with plasmid DNA at various N/P ratios (polymer nitrogen/DNA phosphorus) was investigated by DLS and zeta-potential measurements, indicating that for N/P ≥ 7.5 the complexes bear a positive net charge and do not undergo secondary aggregation. The hybrids were tested as transfection agents under standard conditions in CHO-K1 and L929 cells, revealing transfection efficiencies >50% and low cytotoxicity at N/P ratios of 10 and 15, respectively. Due to the magnetic properties of the hybrid gene vector, it is possible to collect most of the cells that have incorporated a sufficient amount of magnetic material by using a magnetic activated cell sorting system (MACS). Afterward, cells were further cultivated and displayed a transfection efficiency of ca. 60% together with a high viability.
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Separación Celular/métodos , Compuestos Férricos/química , Metacrilatos/química , Nanopartículas/química , Ácidos Polimetacrílicos/síntesis química , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Concentración de Iones de Hidrógeno , Dosificación Letal Mediana , Fenómenos Magnéticos , Ratones , Nanopartículas/toxicidad , Tamaño de la Partícula , Polimerizacion , Ácidos Polimetacrílicos/química , Transfección/métodosRESUMEN
The impact of microplastic particles on organisms is currently intensely researched. Although it is well established that macrophages ingest polystyrene (PS) microparticles, little is known about the subsequent fate of the particles, such as entrapment in organelles, distribution during cell division, as well as possible mechanisms of excretion. Here, submicrometer (0.2 and 0.5 µm) and micron-sized (3 µm) particles were used to analyze particle fate upon ingestion of murine macrophages (J774A.1 and ImKC). Distribution and excretion of PS particles was investigated over cycles of cellular division. The distribution during cell division seems cell-specific upon comparing two different macrophage cell lines, and no apparent active excretion of microplastic particles could be observed. Using polarized cells, M1 polarized macrophages show higher phagocytic activity and particle uptake than M2 polarized ones or M0 cells. While particles with all tested diameters were found in the cytoplasm, submicron particles were additionally co-localized with the endoplasmic reticulum. Further, 0.5 µm particles were occasionally found in endosomes. Our results indicate that a possible reason for the previously described low cytotoxicity upon uptake of pristine PS microparticles by macrophages may be due to the preferential localization in the cytoplasm.
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Microplásticos , Poliestirenos , Animales , Ratones , Poliestirenos/toxicidad , Poliestirenos/metabolismo , Microplásticos/toxicidad , Microplásticos/metabolismo , Plásticos/metabolismo , Macrófagos/metabolismo , Ingestión de AlimentosRESUMEN
Polyelectrolyte microcapsules based on sodium cellulose sulfate (SCS) and poly-diallyl-dimethyl-ammonium chloride (PDADMAC) have previously been proposed as a suitable ex vivo microenvironment for the cultivation and differentiation of primary human T lymphocytes. Here, the same system is investigated for the cultivation of human primary B cells derived from adult tonsillar tissue. Proliferation and differentiation into subtypes are followed and compared to suspension cultures of B cells from the same pool performed in parallel. Total cell expansion is somewhat lower in the capsules than in the suspension cultures. More importantly, however, the differentiation of the initially mainly memory B cells into various subtypes, in particular into plasma cell (PC), shows significant differences. Clearly, the microenvironment provided by the microcapsules is beneficial for an accelerated induction of a germinal center-like B cell phenotype and afterward supports the long-term survival of the PC cells. Then, varying the encapsulation conditions (i.e., presence of human serum and dedicated cytokines in the capsule core) provides a tool for finetuning the B cell response. Hence, this methodology is suggested to pave the way toward ex vivo development of human immune organoids.