Autophagy triggered by reactive oxygen species (ROS) in photodynamic therapy (PDT) generally exhibits an anti-apoptotic effect to promote cell survival. Herein, an innovative supramolecular nanoplatform was fabricated for enhanced PDT by converting the role of autophagy from pro-survival to pro-death. The respiration inhibitor 3-bromopyruvate (3BP), which can act as an autophagy promoter and hypoxia ameliorator, was integrated into photosensitizer chlorin e6 (Ce6)-encapsulated nanoparticles to combat hypoxic tumor. 3BP could inhibit respiration by down-regulating HK-II and GAPDH expression to significantly reduce intracellular oxygen consumption rate, which could relieve tumor hypoxia for enhanced photodynamic cancer therapy. More importantly, the autophagy level was significantly elevated by the combination of 3BP and PDT determined by Western blot, immunofluorescent imaging, and transmission electron microscopy. It was very surprising that excessively activated autophagy promoted cell apoptosis, leading to the changeover of autophagy from pro-survival to pro-death. Therefore, PDT combined with 3BP could achieve efficient cell proliferation inhibition and tumor regression. Furthermore, hypoxia-inducible factor-1α (HIF-1α) could be down-regulated after tumor hypoxia was relieved by 3BP. Tumor metastasis could then be effectively inhibited by eliminating primary tumors and down-regulating HIF-1α expression. These results provide an inspiration for future innovative approaches of cancer therapy by triggering pro-death autophagy.

A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. The resulting peptide analysis has been carried out by matrix-assisted laser desorption ionization timeof-flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayerassembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATR-IR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. The maximum proteolytic velocity of the adsorbed trypsin was ∼600 mM/min µg, thousands of times faster than that in solution. BSA, myoglobin, and cytochrome c were used as model substrates for the tryptic digestion. The standard proteins were identified at a low femtomole per analysis at a concentration of 0.5 ng/µL with the digestion time <5s. This simple technique may offer a potential solution for low-level protein analysis.Progress in the field of biochemistry depends on new analysis techniques that allow studies to be performed on ever-decreasing sample volumes and target analyte concentrations. Many proteins are naturally expressed at low abundance. However, protein analysis at submicromolar concentration or trace levels is inherently difficult, mainly because of low digestion efficiency. Many techniques have been developed to enhance the digestion sensitivity for low levels of proteins. Mann 1 developed digestion of low abundant samples in silver-stained gels. Ramsay 2 reported rapid digestion and analysis of small volumes of proteins using a microchip nanoelectrospray device and TOF spectrometry detection. Li 3,4 proposed a method to digest proteins at low concentrations by a microcolumn filled with enzyme-adsorbing hydrophobic medium. Another alternative procedure is the immobilization of enzymes to digest small sample volumes in nanovials, or capillaries or on chips. Immobilized enzymes are considerably more stable and retain their catalytic activities for much longer time than free enzymes in solution.The microfluidic chip is a potentially powerful tool which has been applied in such diverse research fields as sensors and chemical and biological reactors due to rapid and high-throughput analysis and minimized consumption of sample and reagent, as well as lower manufacturing cost. [5][6][7] Several groups have explored the benefits of immobilized enzymes in microfluidic channels, which would have a significant impact on large-scale protein analysis. Sakai-Kato et al. 8 developed a system consisting of a poly-(methyl methacrylate) with a sample reservoir filled with trypsinencapsulated sol-gel. This on-chip microreactor was applicable to the digestion of protein with multi...

No abstract

No abstract