In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6-8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.
Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.
In this work, we demonstrate a convenient and robust strategy for efficient fabrication of high fluorescence quantum yield (QY, 49.8 ± 3%) semiconducting polymer nanoparticles (SPNs), with size comparable with semiconductor quantum dots (Qdots). The SPNs were synthesized by co-precipitation of hydrophobic semiconducting polymer together with amphiphilic multidentate polymer. Comprehensive spectroscopic and microscopic characterizations showed that the SPNs possess superior photophysical performance, with excellent fluorescence brightness and reduced photoblinking in contrast with Qdots, as well as good photostability compared to a fluorescent protein of a similar size, phycoerythrin. More importantly, by conjugating membrane biomarkers onto the surface of SPNs, it was found that they were not only suitable for specific cellular labelling but also for single particle tracking because of the improved optical performance.
BackgroundInvariant natural killer T cells (iNKT cells) are a unique subset of T lymphocytes and are considered to play an important role in the development of allergic bronchial asthma. Recently, iNKT cells were shown to play an immunoregulatory role in CD4+ and CD8+ T cell-mediated adaptive immune response. Allergen-specific Th2 inflammatory responses are an important part of the adaptive immune response in asthma. However, the regulatory functions of the Th2 inflammatory response in asthma have not been studied in detail.MethodIn this study, we have investigated the regulatory functions of iNKT cells on the Th2 inflammatory response in an ovalbumin (OVA)-induced murine model of asthma.ResultsOur results demonstrate that α-Galactosylceramide (α-GalCer) administration activated iNKT cells but could not induce the Th2 inflammatory response in wild-type (WT) mice. In the OVA-induced asthma model, α-GalCer administration and adoptive transfer of iNKT cells significantly augmented the Th2 inflammatory responses, including elevated inflammatory cell infiltration in the lung and bronchoalveolar lavage fluid (BALF); increased levels of IL-4, IL-5, and IL-13 in the BALF and splenocyte culture supernatant; and increased serum levels of OVA-specific IgE and IgG1. In addition, the Th2 inflammatory response was reduced, but not completely abrogated in CD1d-/- mice immunized and challenged with OVA, compared with WT mice.ConclusionThese results suggest that iNKT cells may serve as an adjuvant to enhance Th2 inflammatory response in an OVA-induced murine model of asthma.
Macrophage phenotype and function varies according to their polarized state, which in turn is dependent on microenvironmental stimuli. Under normal physiological conditions, lung interstitial macrophages that express interleukin (IL)-10 are considered to serve regulatory roles in the prevention of allergic reactions in the airways. However, the phenotypic profile of lung interstitial macrophages during the pathophysiology of asthma remains unknown. In the current study, the phenotypic characteristics of lung interstitial macrophages were investigated in an ovalbumin (OVA)-induced mouse model of asthma. The patterns of surface markers chemokine ligand and interleukin, and the metabolic enzyme activity of lung interstitial macrophages were investigated using flow cytometry analysis, reverse transcription-quantitative polymerase chain reaction, western blot analysis, and ELISA. It was observed that lung interstitial macrophages derived from OVA-induced asthmatic mice expressed phenotypic markers associated with alternatively activated macrophages (M2), including cluster of differentiation-206, transglutaminase 2, arginase (Arg) 1 and chemokine ligand (CCL)17/CCL22/CCL24 secretion. The M2 macrophages also exhibited increased levels of Arg1 activity and reduced levels of IL-10 expression, relative to macrophages derived from control mice. However, when evaluating the expression of markers associated with classically activated (M1) macrophages, namely inducible nitric oxide synthase and IL-12, it was observed that levels of M1 markers in the interstitial macrophages from asthmatic mice did not differ significantly to those in controls. Collectively, these data suggest that lung interstitial macrophages undergo a phenotypic switch from a regulatory macrophage phenotype under normal conditions to an alternative activation state in OVA-induced asthmatic mice.
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