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The cellular protein homeostasis (proteostasis) network responds effectively to insults. In a functional screen in C. elegans, we recently identified the gene receptor-mediated endocytosis 8 (rme-8; human ortholog: DNAJC13) as a component of the proteostasis network. Accumulation of aggregation-prone proteins, such as amyloid-ß 42 (Aß), α-synuclein, or mutant Cu/Zn-superoxide dismutase (SOD1), were aggravated upon the knockdown of rme-8/DNAJC13 in C. elegans and in human cell lines, respectively. DNAJC13 is involved in endosomal protein trafficking and associated with the retromer and the WASH complex. As both complexes have been linked to autophagy, we investigated the role of DNAJC13 in this degradative pathway. In knockdown and overexpression experiments, DNAJC13 acts as a positive modulator of autophagy. In contrast, the overexpression of the Parkinson's disease-associated mutant DNAJC13(N855S) did not enhance autophagy. Reduced DNAJC13 levels affected ATG9A localization at and its transport from the recycling endosome. As a consequence, ATG9A co-localization at LC3B-positive puncta under steady-state and autophagy-induced conditions is impaired. These data demonstrate a novel function of RME-8/DNAJC13 in cellular homeostasis by modulating ATG9A trafficking and autophagy.
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Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Proteostasis , Animales , Caenorhabditis elegans/citología , Células HEK293 , Células HeLa , Humanos , Agregado de ProteínasRESUMEN
We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and growth factors or by the addition of brain pericytes. Under specific conditions expression of angiogenic, angiostatic and neurotrophic factors such as endostatin, pigment epithelium derived factor (PEDF/serpins-F1), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial cell growth factor (VEGF) closely mimicked the in vivo situation. Freeze-fracture analysis of these cultures demonstrated the quality and organization of the endothelial tight junction structures and their association to the two different lipidic leaflets of the membrane. Finally, a multi-cell culture model of the BBB with a transendothelial electrical resistance up to 371 (±15) Ω×cm2 was developed, which may be useful for preliminary screening of drug transport across the BBB and to evaluate cellular crosstalk of cells involved in the neurovascular unit.
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Proteínas Angiogénicas/metabolismo , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Comunicación Celular , Células Endoteliales/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Uniones Estrechas/metabolismo , Animales , Barrera Hematoencefálica/citología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Técnicas de Cocultivo , Impedancia Eléctrica , Humanos , Acoplamiento Neurovascular , Fenotipo , Transducción de Señal , Sus scrofa , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.
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Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Liposomas/metabolismo , Nanopartículas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Línea Celular , Portadores de Fármacos , Humanos , Liposomas/análisis , Liposomas/farmacocinética , Masculino , Nanopartículas/análisis , Péptidos/análisis , Péptidos/farmacocinética , Ratas Wistar , Distribución TisularRESUMEN
BACKGROUND: In general the prediction of the toxicity and therapeutic efficacy of engineered nanoparticles in humans is initially determined using in vitro static cell culture assays. However, such test systems may not be sufficient for testing nanoparticles intended for intravenous application. Once injected, these nanoparticles are caught up in the blood stream in vivo and are therefore in continuous movement. Physical forces such as shear stress and cyclic stretch caused by the pulsatile blood flow are known to change the phenotype of endothelial cells which line the luminal side of the vasculature and thus may be able to affect cell-nanoparticle interactions. METHODS: In this study we investigated the uptake of amorphous silica nanoparticles in primary endothelial cells (HUVEC) cultured under physiological cyclic stretch conditions (1 Hz, 5% stretch) and compared this to cells in a standard static cell culture system. The toxicity of varying concentrations was assessed using cell viability and cytotoxicity studies. Nanoparticles were also characterized for the induction of an inflammatory response. Changes to cell morphology was evaluated in cells by examining actin and PECAM staining patterns and the amounts of nanoparticles taken up under the different culture conditions by evaluation of intracellular fluorescence. The expression profile of 26 stress-related was determined by microarray analysis. RESULTS: The results show that cytotoxicity to endothelial cells caused by silica nanoparticles is not significantly altered under stretch compared to static culture conditions. Nevertheless, cells cultured under stretch internalize fewer nanoparticles. The data indicate that the decrease of nanoparticle content in stretched cells was not due to the induction of cell stress, inflammation processes or an enhanced exocytosis but rather a result of decreased endocytosis. CONCLUSIONS: In conclusion, this study shows that while the toxic impact of silica nanoparticles is not altered by stretch this dynamic model demonstrates altered cellular uptake of nanoparticles under physiologically relevant in vitro cell culture models. In particular for the development of nanoparticles for biomedical applications such improved in vitro cell culture models may play a pivotal role in the reduction of animal experiments and development costs.
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Endocitosis/efectos de los fármacos , Endotelio Vascular/metabolismo , Modelos Biológicos , Nanopartículas/metabolismo , Dióxido de Silicio/metabolismo , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Exocitosis/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Cinética , Nanopartículas/toxicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de la Partícula , Flujo Pulsátil , Dióxido de Silicio/toxicidad , Propiedades de SuperficieRESUMEN
In the manufacturing process of ship propellers, large quantities of grinding chips are generated. These grinding chips result from the finishing of the blade surfaces after the primary casting process of the propeller. The aim of this study was to investigate and compare different preparation processes used to produce chip powders with sufficient powder quality for the additive manufacturing process of directed energy deposition. The preparation of the samples was performed through different sieving, milling and re-melting processes. For the characterization of the prepared samples, powder analysis according to relevant industry standards was carried out. It was found that the re-melting processes result in superior powder quality for additive manufacturing in terms of particle size, morphology, and flowability. For some characteristics, the powder exhibits even better properties than those of commercial powders. Furthermore, the powder properties of the milled samples demonstrate a promising potential for use in additive manufacturing.
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A library-orientated approach is used to gain understanding of the interactions of well-defined nanoparticles with primary human endothelial cells, which are a key component of the vasculature. Fifteen sequentially modified gold nanoparticles (AuNPs) based on three different core sizes (18, 35, 65 nm) and five polymeric coatings were prepared. The synthetic methodology ensured homogeneity across each series of particles to allow sequential investigation of the chemical features on cellular interactions. The toxicity of these nanoparticles, their uptake behavior in primary human dermal microvascular endothelial cells (HDMECs), and quantification of uptake were all investigated. The results of our studies indicated that high concentrations of gold nanoparticles (250 µg/mL) were nontoxic and that the number of internalized nanoparticles was related to nanoparticle size and surface chemistry. In summary, the positive-charged ethanediamine-coated AuNPs were internalized to a greater extent than the negative- or neutral-charged AuNPs. Moreover, differences in the amounts of internalized AuNPs could be shown for the three neutral-charged AuNPs, whereas the uptake of hydroxypropylamine-coated particles was preferred compared with glucosamine-coated or PEGylated AuNPs. Hydroxypropylamine-coated AuNPs were found to be the most efficient neutral-charged particles in overcoming the endothelial cell barrier and entering the cell.
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Células Endoteliales/química , Oro/química , Nanopartículas del Metal/química , Microvasos/química , Polímeros/química , Piel/química , Supervivencia Celular , Células Cultivadas , Células Endoteliales/citología , Etilenodiaminas/química , Glucosamina/química , Humanos , Microvasos/citología , Tamaño de la Partícula , Piel/citología , Propiedades de SuperficieRESUMEN
BACKGROUND: The use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm), to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier. RESULTS: Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm), the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the nanoparticles' surface. CONCLUSIONS: In conclusion the higher amount of citrate on the particle surface resulted in a higher impairment of cell viability, but did not enhance or reduce the uptake behavior in endothelial or epithelial cells. In addition, epithelial and endothelial cells exhibited different uptake behaviors for citrate-stabilized gold nanoparticles, which might be related to different interactions occurring at the nanoparticle-cell-surface interface. The different uptake in epithelial cells might explain the higher reduction of proliferation of these cells after exposure to AuNPs treatment although more detailed investigations are necessary to determine subcellular events. Nevertheless an extrinsic effect of sodium-citrate stabilized particles could not be excluded. Thus, the amount of sodium citrate should be reduced to a level on which the stability of the particles and the safety for biomedical applications are guaranteed.
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Citratos/toxicidad , Materiales Biocompatibles Revestidos/toxicidad , Endotelio Vascular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Nanosferas/toxicidad , Barrera Hematoencefálica/citología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citratos/química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Prepucio/citología , Oro/química , Oro/metabolismo , Humanos , Masculino , Nanopartículas del Metal/química , Microscopía Electrónica de Transmisión , Nanosferas/química , Tamaño de la Partícula , Citrato de SodioRESUMEN
All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.
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Detailed examination of tumor components is leading-edge to establish personalized cancer therapy. Accompanying research on cell-free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter- and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully-automated enrichment and single cell dispensing of CTCs from whole blood without pre-processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR-based confirmation of tumor-related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real-time monitoring.
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The blood-brain barrier (BBB) maintains the homeostasis of the central nervous system, which is one of the reasons for the treatments of brain disorders being challenging in nature. Nanoparticles (NPs) have been seen as potential drug delivery systems to the brain overcoming the tight barrier of endothelial cells. Using a BBB model system based on human induced pluripotent stem cells (iPSCs), the impact of polymeric nanoparticles has been studied in relation to nanoparticle size, material, and protein corona. PLGA [poly(lactic-co-glycolic acid)] and PLLA [poly(d,l-lactide)] nanoparticles stabilized with Tween® 80 were synthesized (50 and 100 nm). iPSCs were differentiated into human brain microvascular endothelial cells (hBMECs), which express prominent BBB features, and a tight barrier was established with a high transendothelial electrical resistance of up to 4000 Ω cm2. The selective adsorption of proteins on the PLGA and PLLA nanoparticles resulted in a high percentage of apolipoproteins and complement components. In contrast to the prominently used BBB models based on animal or human cell lines, the present study demonstrates that the iPSC-based model is suited to study interactions with nanoparticles in correlation with their material, size, and protein corona composition. Furthermore, asymmetrical flow field-flow fractionation enables the investigation of size and agglomeration state of NPs in biological relevant media. Even though a similar composition of the protein corona has been detected on NP surfaces by mass spectrometry, and even though similar amounts of NP are interacting with hBMECs, 100 nm-sized PLGA NPs do impact the barrier, forming endothelial cells in an undiscovered manner.
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Barrera Hematoencefálica/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Nanopartículas/química , Tamaño de la Partícula , Polímeros/química , Corona de Proteínas/química , Calibración , Diferenciación Celular , Dispersión Dinámica de Luz , Impedancia Eléctrica , Células Endoteliales/metabolismo , Fraccionamiento de Campo-Flujo , Humanos , Nanopartículas/toxicidad , Nanopartículas/ultraestructura , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Poliestirenos/química , Electricidad EstáticaRESUMEN
BACKGROUND: detailed information about circulating tumor cells (CTCs) as an indicator of therapy response and cancer metastasis is crucial not only for basic research but also for diagnostics and therapeutic approaches. Here, we showcase a newly developed IsoMAG IMS system with an optimized protocol for fully automated immunomagnetic enrichment of CTCs, also revealing rare CTC subpopulations. METHODS: using different squamous cell carcinoma cell lines, we developed an isolation protocol exploiting highly efficient EpCAM-targeting magnetic beads for automated CTC enrichment by the IsoMAG IMS system. By FACS analysis, we analyzed white blood contamination usually preventing further downstream analysis of enriched cells. RESULTS: 1 µm magnetic beads with tosyl-activated hydrophobic surface properties were found to be optimal for automated CTC enrichment. More than 86.5% and 95% of spiked cancer cells were recovered from both cell culture media or human blood employing our developed protocol. In addition, contamination with white blood cells was minimized to about 1200 cells starting from 7.5 mL blood. Finally, we showed that the system is applicable for HNSCC patient samples and characterized isolated CTCs by immunostaining using a panel of tumor markers. CONCLUSION: Herein, we demonstrate that the IsoMAG system allows the detection and isolation of CTCs from HNSCC patient blood for disease monitoring in a fully-automated process with a significant leukocyte count reduction. Future developments seek to integrate the IsoMAG IMS system into an automated microfluidic-based isolation workflow to further facilitate single CTC detection also in clinical routine.
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Micromixer technology was used to manufacture magnetic single core iron oxide nanoparticles that combine imaging as well as therapeutic functions. In a continuous, scalable and highly controllable manner, synthesis with biocompatible educts via an aqueous synthesis route was performed. Size control by varying relevant process parameters e.g. temperature was confirmed by transmission electron microscopy measurements of experimental series demonstrating the exceptional size control and homogeneity. Furthermore, analytical centrifugation evidenced the stably dispersed state of the single core nanoparticles in aqueous media. Size controlled production of single-core iron oxide nanoparticles was used to design optimized nanoparticles with a core diameter of about 30 nm, showing high signal amplitudes in Magnetic Particle Imaging (MPI) as a promising MPI tracer material. Moreover, therapeutic potential of these particles in magnetic fluid hyperthermia was evaluated and specific absorption rates (SAR values) up to 1 kW per g(Fe) were obtained, which exceed the comparable SAR value of Resovist® by more than a factor of three. Relaxometry measurements clearly confirmed the capacity of these single-core magnetic nanoparticles to generate significant T 2-weighted magnetic resonance imaging (MRI) contrast that potentially allows multimodal imaging for monitoring the particles in vivo in a theranostic application scenario. Finally, first cell viability and apoptosis tests on endothelial cells did not show any cytotoxicity certifying a good biocompatibility of the iron oxide nanoparticles. This microtechnological approach provides reproducible, scalable single core iron oxide nanoparticles as highly performing tracers for MPI diagnosis as well as efficient heat generators for hyperthermia therapy. These preliminary results contribute to translational research in image guided cancer therapy - a further step from basic research to future medicine.
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A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood-brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modifications) for various times and evaluated their effects on cytotoxicity, proinflammatory mediators, barrier functions and factors involved in ER stress. We demonstrated a time-dependent uptake of all AuNPs and no cytotoxicity for up to 72 h of exposure. Exposure to certain AuNPs resulted in a time-dependent increase in the proinflammatory markers IL-8, MCP-1, sVCAM, sICAM. However, none of the AuNPs induced an increase in expression of the chaperones and stress sensor proteins BiP and GRP94, respectively, or the transcription factors ATF4 and ATF6. Furthermore, no upregulation of the UPR stress sensor receptor PERK, no active splicing product of the transcription factor XBP1 and no upregulation of the transcription factor CHOP were detectable. In conclusion, the results of the present study indicate that effects of different-sized gold nanoparticles modified with various polymers were not related to the induction of ER stress in brain microvascular endothelial cells or led to apoptosis.
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Barrera Hematoencefálica/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Polímeros/química , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células Endoteliales/metabolismo , Oro/química , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Interleucina-8/metabolismo , Proteínas de la Membrana/metabolismo , Nanopartículas del Metal/química , Tamaño de la Partícula , Factores de Riesgo , Factor de Transcripción CHOP/metabolismoRESUMEN
Transporters of the ATP-binding cassette (ABC) family such as MDR1 play a pivotal role in persistence of brain homeostasis by contributing to the strict permeability properties of the blood-brain barrier. This barrier on one hand compromises treatment of central nervous system diseases by restricting access of drugs; on the other hand, an impaired or altered function of barrier building cells has been described in neurological disorders. The latter might contribute to increased vulnerability of the brain under pathological conditions or even enforce pathogenesis. Here, we present a novel approach for a systematic examination of drug impact on Mdr1 gene expression by establishing a dual reporter gene assay for the murine upstream core promoters of Mdr1a and b. We validated the time-resolved assay in comparison with single reporter gene constructs and applied it to analyze effects of a Food and Drug Administration (FDA)-approved drug library consisting of 627 substances. The chemo-preventive synthetic dithiolethione oltipraz was reidentified with our assay as an already known inducer of Mdr1 gene expression. Together with two newly characterized modifiers - gemcitabine and trichlormethiazide - we prove our findings in a blood-brain barrier culture model as well as in wild-type and Mdr1 knockout mice. In sum, we could demonstrate that our dual reporter gene assay delivers results, which also persist in the living animal and consequently is applicable for further analysis and prediction of Mdr1 regulation in vivo.
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Responsive, theranostic nanosystems, capable of both signaling and treating wound infections, is a sophisticated approach to reduce the most common and potentially traumatizing side effects of burn wound treatment: slowed wound healing due to prophylactic anti-infective drug exposure as well as frequent painful dressing changes. Antimicrobials as well as dye molecules have been incorporated into biodegradable nanosystems that release their content only in the presence of pathogens. Following nanocarrier degradation by bacterial enzymes, any infection will thus emit a visible signal and be effectively treated at its source. In this study, we investigated the effect of fluorescent-labeled hyaluronan nanocapsules containing polyhexanide biguanide and poly-L-lactic acid nanoparticles loaded with octenidine on primary human dermal microvascular endothelial cells, which play a major role in cutaneous wound healing. Microscopic and flow cytometric analysis indicated a time-dependent uptake of both the nanocapsules and the nanoparticles. However, enzyme immunoassays showed no significant influence on the expression of pro-inflammatory cell adhesion molecules and cytokines by the endothelial cells. Under angiogenic-stimulating conditions, the potential to form capillary-like structures in co-culture with dermal fibroblasts was not inhibited. Furthermore, cytotoxicity studies (the MTS and crystal violet assay) after short- and long-term exposure to the materials demonstrated that both systems exhibited less toxicity than solutions of the antiseptic agents alone in comparable concentrations. The results indicate that responsive antimicrobial nanocomposites could be used as an advanced drug delivery system and a promising addition to current best practice wound infection prophylaxis with few side effects.
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Quemaduras/tratamiento farmacológico , Nanocompuestos/uso terapéutico , Cicatrización de Heridas , Infección de Heridas/tratamiento farmacológico , Células Cultivadas , Fibroblastos , Humanos , Modelos BiológicosRESUMEN
BACKGROUND: Alzheimer's disease represents one of the main neurological disorders in the aging population. Treatment options so far are only of symptomatic nature and efforts in developing disease modifying drugs by targeting amyloid beta peptide-generating enzymes remain fruitless in the majority of human studies. During the last years, an alternative approach emerged to target the physiological alpha-secretase ADAM10, which is not only able to prevent formation of toxic amyloid beta peptides but also provides a neuroprotective fragment of the amyloid precursor protein - sAPPalpha. PURPOSE: To identify novel alpha-secretase enhancers from a library of 313 extracts of medicinal plants indigenous to Korea, a screening approach was used and hits were further evaluated for their therapeutic value. METHODS: The extract library was screened for selective enhancers of ADAM10 gene expression using a luciferase-based promoter reporter gene assay in the human neuroblastoma cell line SH-SY5Y. Candidate extracts were then tested in wild type mice for acute behavioral effects using an open field paradigm. Brain and liver tissue from treated mice was biochemically analyzed for ADAM10 gene expression in vivo. An in vitro blood-brain barrier model and an in vitro ATPase assay were used to unravel transport properties of bioactive compounds from extract candidates. Finally, fractionation of the most promising extract was performed to identify biologically active components. RESULTS: The extract of Caragana sinica (Buc'hoz) Rehder was identified as the best candidate from our screening approach. We were able to demonstrate that the extract is acutely applicable in mice without obvious side effects and induces ADAM10 gene expression in peripheral tissue. A hindered passage across the blood-brain barrier was detected explaining lack of cerebral induction of ADAM10 gene expression in treated mice. By fractionating C. sinica extract we identified alpha-viniferin as one of the biologically active components. CONCLUSION: The extract of C. sinica and alpha-viniferin as one of its bioactive constituents might serve as novel therapeutic options for treating Alzheimer's disease by increasing ADAM10 gene expression. The identification of alpha-viniferin represents a promising starting point to achieve blood-brain barrier penetrance in the future.
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Caragana/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/agonistas , Extractos Vegetales/farmacología , Plantas Medicinales/química , Proteínas ADAM , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Benzofuranos/química , Benzofuranos/farmacología , Barrera Hematoencefálica , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Noqueados , Extractos Vegetales/química , Regiones Promotoras GenéticasRESUMEN
In the pathogenesis of Alzheimer's disease (AD) the homeostasis of amyloid precursor protein (APP) processing in the brain is impaired. The expression of the competing proteases ADAM10 (a disintegrin and metalloproteinase 10) and BACE-1 (beta site APP cleaving enzyme 1) is shifted in favor of the A-beta generating enzyme BACE-1. Acitretin--a synthetic retinoid-e.g., has been shown to increase ADAM10 gene expression, resulting in a decreased level of A-beta peptides within the brain of AD model mice and thus is of possible value for AD therapy. A striking challenge in evaluating novel therapeutically applicable drugs is the analysis of their potential to overcome the blood-brain barrier (BBB) for central nervous system targeting. In this study, we established a novel cell-based bio-assay model to test ADAM10-inducing drugs for their ability to cross the BBB. We therefore used primary porcine brain endothelial cells (PBECs) and human neuroblastoma cells (SH-SY5Y) transfected with an ADAM10-promoter luciferase reporter vector in an indirect co-culture system. Acitretin served as a model substance that crosses the BBB and induces ADAM10 expression. We ensured that ADAM10-dependent constitutive APP metabolism in the neuronal cells was unaffected under co-cultivation conditions. Barrier properties established by PBECs were augmented by co-cultivation with SH-SY5Y cells and they remained stable during the treatment with acitretin as demonstrated by electrical resistance measurement and permeability-coefficient determination. As a consequence of transcellular acitretin transport measured by HPLC, the activity of the ADAM10-promoter reporter gene was significantly increased in co-cultured neuronal cells as compared to vehicle-treated controls. In the present study, we provide a new bio-assay system relevant for the study of drug targeting of AD. This bio-assay can easily be adapted to analyze other Alzheimer- or CNS disease-relevant targets in neuronal cells, as their therapeutical potential also depends on the ability to penetrate the BBB.
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Acitretina/farmacología , Bioensayo , Células Endoteliales/efectos de los fármacos , Queratolíticos/farmacología , Neuronas/efectos de los fármacos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Acitretina/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Transporte Biológico , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Sistemas de Liberación de Medicamentos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Queratolíticos/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Neuronas/citología , Neuronas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , PorcinosRESUMEN
Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product.
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Endotelio Vascular/citología , Endotoxinas/análisis , Cangrejos Herradura/química , Animales , Células Cultivadas , Humanos , Límite de DetecciónRESUMEN
Poly(amidoamine)s with bioreducible disulfide linkages in the main chain (SS-PAAs) and pH-responsive, negatively charged citraconate groups in the sidechain have been designed for effective intracellular delivery and release of proteins with a net positive charge at neutral pH. Using lysozyme as a cationic model protein these water soluble polymers efficiently self-assemble into nanocomplexes by charge attraction. At pH5 (the endosomal pH) the amide linkages connecting the citraconate groups in the sidechains of the SS-PAAs are hydrolyzed by intramolecular catalysis, resulting in expulsion of the negative citraconate groups and formation of protonated amine groups, resulting in charge reversal of the polymeric carrier from negative to positive. The concomitant endosomal buffering effect and increased polymer-endosomal membrane interactions are considered to lead to increased protein delivery into the cytosol. Besides destabilization of the polymer-protein nanoparticles by the charge reversal effect, intracellular cleavage of disulfide linkages in the polymer ensure further unpacking of the protein in the cytosol. Cellinternalization and cytotoxicity experiments with primary human umbilical vein endothelial cells (HUVEC) showed that the SS-PAA-based nanocomplexes were essentially non-toxic, and that lysozyme is successfully internalized into HUVEC. The results indicate that these charge reversal SS-PAAs have excellent properties as non-toxic intracellular delivery systems for cationic proteins.
Asunto(s)
Células Endoteliales/química , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Poliaminas/química , Proteínas/administración & dosificación , Proteínas/química , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Humanos , Ensayo de MaterialesRESUMEN
The facile and modular functionalization of gold nanoparticles makes them versatile tools in nanomedicine, for instance, photothermal therapy, contrast agents or as model nanoparticles to probe drug-delivery mechanisms. Since endothelial cells from various locations in the body exhibit unique phenotypes we quantitatively examined the amount of different sized poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles internalized into primary human dermal endothelial cells or human brain endothelial cells (hCMEC/D3) by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and visualized the nanoparticles using light and electron microscopy. Poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles exhibited high uptake into brain endothelial cells and were used to examine transport mechanisms across the blood-brain barrier using a well-established in vitro model of the blood-brain barrier. Our results demonstrate that 35 nm-sized gold nanoparticles were internalized best into human brain endothelial cells by a flotillin-dependent endocytotic pathway. The uptake into the cells is not correlated with transport across the blood-brain barrier. We demonstrated that the surface modification of gold nanoparticles impacts the internalization process in different cells. In addition, to evaluating toxicity and uptake potential of nanoparticles into cells, the transport properties across cell barriers are important criteria to classify nanoparticle properties regarding targeted delivery of drugs.