RESUMO
We develop a droplet microfluidic platform to increase the concentration of analytes in solution via reduction of the sample volume under well-defined conditions. This approach improves the detection and quantification of analytes without requiring any a priori information on their structure nor physical chemical properties. Samples are compartmentalized and processed in water-in-oil droplets that are individually stored in cylindrical microwells located on top of a microfluidic channel. The individual droplets shrink over time due to water extraction in the surrounding oil, leading to an increase in the analyte concentration up to 100,000-fold within the droplet. We demonstrate the power of this approach for detection applications by quantifying a broad range of single analytes such as small molecules, proteins, nanoparticles, exosomes, and amyloid fibrils. With this setup, we can measure pM concentrations, corresponding to zeptomole (10-21 mol) amounts encapsulated in individual droplets. We further show that the droplet concentrator device, or DroMiCo, can quantify unlabeled proteins in nM concentrations and analyze multicomponent mixtures when coupled with a prefractionation step. We illustrate this concept by detecting femtomoles (10-15 mol) of soluble protein oligomers prefractionated by size exclusion chromatography. Finally, we apply the DroMiCo to the analysis of phase diagrams of macromolecules, including synthetic polymers and proteins. Specifically, we analyze the liquid-liquid phase separation of an in vitro model of cellular membraneless compartments, composed of a phase separating protein in the presence of defined concentrations of molecular modulators such as RNA and ATP.
Assuntos
Técnicas Analíticas Microfluídicas , Proteínas/análise , Tamanho da Partícula , Soluções , Propriedades de SuperfícieRESUMO
Combination chemotherapy has proven to be a favorable strategy to treat acute leukemia. However, the introduction of novel compounds remains challenging and is hindered by a lack of understanding of their mechanistic interactions with established drugs. In the present study, we demonstrate a highly increased response of various acute leukemia cell lines, drug-resistant cells and patient-derived xenograft cells by combining the recently introduced protein disulfide isomerase inhibitor PS89 with cytostatics. In leukemic cells, a proteomics-based target fishing approach revealed that PS89 affects a whole network of endoplasmic reticulum homeostasis proteins. We elucidate that the strong induction of apoptosis in combination with cytostatics is orchestrated by the PS89 target B-cell receptor-associated protein 31, which transduces apoptosis signals at the endoplasmic reticulum -mitochondria interface. Activation of caspase-8 and cleavage of B-cell receptor-associated protein 31 stimulate a pro-apoptotic crosstalk including release of calcium from the endoplasmic reticulum and an increase in the levels of reactive oxygen species resulting in amplification of mitochondrial apoptosis. The findings of this study promote PS89 as a novel chemosensitizing agent for the treatment of acute leukemia and uncovers that targeting the endoplasmic reticulum - mitochondrial network of cell death is a promising approach in combination therapy.
Assuntos
Citostáticos/farmacologia , Retículo Endoplasmático/metabolismo , Leucemia/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Leucemia/patologia , Camundongos , Modelos Biológicos , Proteoma , Proteômica/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated ß-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.
Assuntos
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Queratinócitos/metabolismo , Morte Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismoRESUMO
Their natural functions in intercellular communication render extracellular vesicles (EV) highly attractive for drug delivery applications. However, the loading efficiency of present methods to incorporate particularly hydrophilic low molecular weight drugs of biomedical interest is largely unexplored, as is the impact these methods may have on the intrinsic structural and biological vesicle properties. Here, different methods are exploited to incorporate hydrophilic non-membrane permeable compounds into stem cell-derived small EV, and to assess the vesicle characteristics after the different loading processes. When comparing several methods head-to-head, the loading capacity increases in the order saponin ≤ sonication < fusion < freeze-thawing ≤ osmotic shock. Interestingly, the structural and biological functions of small EV are dependent on the applied encapsulation process, with the functional properties being altered at a greater extent. Therefore, the importance of including additional characterization parameters to probe alterations of the biological functionality of small EV is clearly demonstrated. Here, freeze-thawing and particularly the osmotic shock have proven to be the most appropriate methods for EV loading, as they achieve a high drug encapsulation and yet preserve the investigated structural and biological vesicle characteristics.
Assuntos
Vesículas Extracelulares , Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/química , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , SonicaçãoRESUMO
Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.
Assuntos
Vesículas Extracelulares , Microfluídica , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Dispositivos Lab-On-A-Chip , Reprodutibilidade dos TestesRESUMO
Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes' biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV-16 E6/E7 transformed human bone marrow stromal cell-derived exosomes are identified as key bioactive components promoting the exosome-driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes' biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type.