Designer exosomes enabling tumor targeted efficient chemo/gene/photothermal therapy.

Exosomes, endogenous nanosized particles (50-150 nm) secreted and absorbed by cells, have been recently used as diagnostic and therapeutic platforms in cancer treatment. The integration of exosome-based delivery with multiple therapeutic modalities could result in better clinical outcomes and reduced-sided effects. Here, we combined the targeting and biocompatibility of designer exosomes with chemo/gene/photothermal therapy. Our platform consists of exosomes loaded with internalized doxorubicin (DOX, a model cancer drug) and coated with magnetic nanoparticles conjugated with molecular beacons capable of targeting miR-21 for responsive molecular imaging. The coated magnetic nanoparticle enables enrichment of the exosomes at the tumor site by external magnetic field guidance. After the exosomes are gathered at the tumor site, the application of near-infrared radiation (NIR) induces localized hyperthermia and triggers the release of cargoes loaded inside the exosome. The released molecular beacon can target the miR-21 for both imaging and gene silencing. Meanwhile, the released doxorubicin serves to kill the cancer cells. About 91.04 % of cancer cells are killed after treatment with Exo-DOX-Fe3O4@PDA-MB under NIR. The ability of the exosome-based method for cancer therapy has been demonstrated by animal models, in which the tumor size is reduced dramatically by 97.57 % with a magnetic field-guided tumor-targeted chemo/gene/photothermal approach. Thus, we expected this designer exosome-mediated multi-mode therapy to be a promising platform for the next-generation precision cancer nanomedicines.

A review on microfluidics manipulation of the extracellular chemical microenvironment and its emerging application to cell analysis.

Spatiotemporal manipulation of extracellular chemical environments with simultaneous monitoring of cellular responses plays an essential role in exploring fundamental biological processes and expands our understanding of underlying mechanisms. Despite the rapid progress and promising successes in manipulation strategies, many challenges remain due to the small size of cells and the rapid diffusion of chemical molecules. Fortunately, emerging microfluidic technology has become a powerful approach for precisely controlling the extracellular chemical microenvironment, which benefits from its integration capacity, automation, and high-throughput capability, as well as its high resolution down to submicron. Here, we summarize recent advances in microfluidics manipulation of the extracellular chemical microenvironment, including the following aspects: i) Spatial manipulation of chemical microenvironments realized by convection flow-, diffusion-, and droplet-based microfluidics, and surface chemical modification; ii) Temporal manipulation of chemical microenvironments enabled by flow switching/shifting, moving/flowing cells across laminar flows, integrated microvalves/pumps, and droplet manipulation; iii) Spatiotemporal manipulation of chemical microenvironments implemented by a coupling strategy and open-space microfluidics; and iv) High-throughput manipulation of chemical microenvironments. Finally, we briefly present typical applications of the above-mentioned technical advances in cell-based analyses including cell migration, cell signaling, cell differentiation, multicellular analysis, and drug screening. We further discuss the future improvement of microfluidics manipulation of extracellular chemical microenvironments to fulfill the needs of biological and biomedical research and applications.

Activating Antitumor Immunity and Antimetastatic Effect Through Polydopamine-Encapsulated Core-Shell Upconversion Nanoparticles.

Synergistic phototherapy has the potential to conquer the extreme heterogeneity and complexity of difficult tumors and result in better cancer treatment outcomes than monomodal photodynamic therapy (PDT) or photothermal therapy (PTT). However, the previous approaches to combining PDT and PTT are mainly focused on primary tumor obliteration while neglecting tumor metastasis, which is responsible for about 90% of cancer deaths. It is shown that a combined PDT/PTT approach, based on upconversion-polymer hybrid nanoparticles with surface-loaded chlorin e6 photosensitizer, can enhance primary tumor elimination and elicit antitumor immunity against disseminated tumors. The specifical arrangement of an external upconversion coating over the polymer core ensures adequate photoabsorption by the upconversion nanoparticles for the generation of reactive oxygen species upon single near-infrared light irradiation. Furthermore, it is found that synergistic phototherapy can elicit robust systemic and humoral antitumor immune responses. When combined with immune checkpoint blockades, it can inhibit tumor relapse and metastasis as well as prolong the survival of tumor-bearing mice in two types of tumor metastasis models. This study may establish a new modality for enhancing immunogenic cell death through a synergistic phototherapeutic nanoplatform and extend this strategy to overcome tumor metastasis with an augmented antitumor immune response.

Real-time monitoring of immune responses under pathogen invasion and drug interference by integrated microfluidic device coupled with worm-based biosensor.

Immune response to environmental pathogen invasion is a complex process to prevent host from further damage. For quantitatively understanding immune responses and revealing the pathogenic environmental information, real-time monitoring of such a whole dynamic process with single-animal resolution in well-defined environments is highly desired. In this work, an integrated microfluidic device coupled with worm-based biosensor was proposed for in vivo studies of dynamic immune responses and antibiotics interference in infected C. elegans. Individual worms housed in chambers were exposed to the various pathogens and discontinuously manipulated for imaging with limited influence on physiological activities. The expression of immune responses gene (irg-1) was time-lapse measured in intact worms during pathogen infection. Results demonstrated that irg-1 gene could be induced in the presence of P. aeruginosa strain PA14 in a dose-dependent manner, and the survival of infected worm could be rescued under gentamicin or erythromycin treatments. We expect it to be a versatile platform to facilitate future studies on pathogenesis researches and rapid drug screen using C. elegans disease model.

Synthesis and Biological Evaluation of Novel σ1 Receptor Ligands for Treating Neuropathic Pain: 6-Hydroxypyridazinones.

By use of the 6-hydroxypyridazinone framework, a new series of potent σ1 receptor ligands associated with pharmacological antineuropathic pain activity was synthesized and is described in this article. In vitro receptor binding studies revealed high σ1 receptor affinity (Ki σ1 = 1.4 nM) and excellent selectivity over not only σ2 receptor (1366-fold) but also other CNS targets (adrenergic, µ-opioid, sertonerigic receptors, etc.) for 2-(3,4-dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)pyridazin-3(2H)-one (compound 54). Compound 54 exhibited dose-dependent antiallodynic properties in mouse formalin model and rats chronic constriction injury (CCI) model of neuropathic pain. In addition, functional activity of compound 54 was evaluated using phenytoin and indicated that the compound was a σ1 receptor antagonist. Moreover, no motor impairments were found in rotarod tests at antiallodynic doses and no sedative side effect was evident in locomotor activity tests. Last but not least, good safety and favorable pharmacokinetic properties were also noted. These profiles suggest that compound 54 may be a member of a novel class of candidate drugs for treatment of neuropathic pain.

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis.

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated.

Separation and determination of amino acids by micellar electrokinetic chromatography coupling with novel multiphoton excited fluorescence detection.

In this article, it was demonstrated that separation and determination of 20 amino acids were accomplished by micellar electrokinetic chromatography (MEKC) coupling with novel multiphoton excited fluorescence (MPEF) detection method. Different from MPEF achieved by expensive fs laser, continuous wave (CW) diode laser of ultra-low cost was uniquely employed in our MPEF system. Amino acids were fluorescently labeled with fluorescein isothiocyanate (FITC), and were subjected to sodium dodecyl sulfate (SDS)-based MEKC separation and CW-based MPEF detection. The result was compared with that by single photon excited fluorescence (SPEF), which indicated that MPEF had the advantages of better mass detectability and higher separation selectivity over SPEF. Quantitative analysis was performed and revealed linear dynamic range of over 2 orders of magnitude, with mass detection limit down to ymole level. To evaluate the reliability, this method was successfully applied for analyzing a commercial nutrition supplement liquid.

Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice.

A Ca2+/calmodulin (CaM)-binding protein kinase from rice ( Oryza sativa ), OsCBK, has been characterized that lacks Ca2+-binding EF hands and has Ca2+/CaM-independent autophosphorylation and substrate-phosphorylation activity. OsCBK has all 11 subdomains of a kinase catalytic domain and a putative CaM-binding domain, and shares high identity with Ca2+-dependent-protein-kinase ('CDPK')-related protein kinases in plants. OsCBK bound CaM in a Ca2+-dependent manner as previously reported for Ca2+/calmodulin-dependent protein kinases in animals, but autophosphorylation and phosphorylation of histone IIIs were Ca2+/CaM-independent. Surface plasmon resonance analysis showed that OsCBK specifically bound CaM with high affinity ( K (D)=30 nM). Capillary electrophoresis showed that phosphorylation of OsCBK occurred on serine and threonine residues. These data show that OsCBK is a serine/threonine protein kinase that binds Ca2+/CaM, but whose enzymic activity is independent of Ca2+/CaM. In situ hybridization showed that OsCBK is expressed in reproductive and vegetative tissues of rice and shows temporal and spatial changes during plant growth and development. OsCBK is highly expressed in zones of cell division and it is particularly abundant in sporogenous cells of the anther at meiosis.