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We developed a multiplex single-molecule quantitative assay of intracellular telomerase that used target-triggered signal amplification to enhance sensitivity, substrate reaction to increase signal stability, and quantum dots to enhance signal-to-noise ratio, obtaining an LOD of 5 × 10-14 IU for intracellular telomerase and LOD of 3 cells for multiple cancer cells.

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DNA nanomachines could initiate the cascade reaction in an autonomous mode under the drive of triggers, which achieve the signal amplification for the bioimaging of intracellular biomarkers. Compared with the "always-on" nanomachine that possibly produces false-positive signals, a controllable nanomachine with the on-site activation could be better for accurate tumor imaging and precise tumor therapy. Till now, the endogenous and exogenous triggers have been developed to design the controllable nanosensors. However, their combinations to develop feasible DNA nanomachines have been rarely studied. Herein, we constructed a near-infrared (NIR)-light-controlled DNA nanomachine that was first activated by the NIR light and then induced a target-triggered amplification process under the drive of an endogenous stimulus. Owing to adenosine-5'-triphosphate (ATP) having much higher concentration in cancer cells than that in healthy cells and the extracellular fluid, the obtained DNA nanomachine was selectively activated in cancer cells with inhibited interference signals from the surrounding healthy tissues. With obvious advantages including the exogenous NIR light initiation, the selective activation by the target microRNA, and the sensitive acceleration by the ATP-induced strand recycling reaction, the constructed nanomachine could be used to image the intracellular microRNA with increased sensitivity. Besides, after modifying the DNA sequence with the photosensitizer molecules, the obtained nanomachine could perform the selective photodynamic therapy on the tumor sections with the outstandingly decreased side effects. Thus, we hope the designed nanomachine could provide some important hints to design feasible nanomachines for accurate tumor diagnosis and precise tumor therapy.

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The construction of a sensitive strategy for in situ visualizing and dynamic tracing intracellular microRNA is of great importance. Via the toehold-mediated strand displacement process, the catalytic hairpin assembly (CHA) could offer several hundreds-fold signal amplifications. However, the CHA may produce certain background interferences since microRNA may exist in normal cells. In this work, we constructed an endogenously and sequentially activated signal amplification strategy to provide the amplified dual-color fluorescence imaging of microRNA in living cancer cells, which was comprised of two successive reaction processes: the activation of the preprotective catalytic probe by the endogenous glutathione (GSH) and the subsequent catalytic hairpin assembly on the surface of the upconversion nanoprobe triggered by the specific microRNA. Since the concentration of GSH in cancer cells was much higher than that in normal cells and the extracellular environment, the activation of the designed nanoprobe could be controlled at the desirable site. With the merits of the endogenous initiation and selective activation, the designed nanoprobe could achieve the bioimaging of microRNA in living cancer cells with high precision and reliability. Furthermore, via the introduction of a photosensitizer molecule into the DNA strand, the designed nanoplatform could achieve the precise photodynamic therapy (PDT) for cancer cells and malignant tumors under the irradiation of the NIR laser. This work provided a new avenue to achieve the accurate imaging of intracellular microRNA and guided precise PDT, which would offer powerful hints to the early diagnosis and therapy of malignant tumors.

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Most photosensitizer molecules used for the photodynamic therapy (PDT) are chemically-synthesized organic photosensitizer dyes which show several limitations such as unsatisfactory cell uptake, weak selectivity and off-target phototoxicity. Recently, genetically-encoded photosensitizers have attracted increasing attentions which provide the targeted cell elimination with single-cell precision. However, their applications are mainly limited by the shallow tissue penetration depth of the excitation light and the low cell apoptosis ratio. Herein, we developed a feasible upconversion nanoparticle (UCNP)-based optogenetic nanosystem with three-in-one functional integration: bio-imaging, NIR-triggered PDT and cascade gene therapy. Firstly, the mitochondria-targeted genetically-encoded photosensitizer was constructed and transfected into cancer cells. Then, the functional upconversion nanoprobe was constructed with the mitochondria targetability and then the siRNA was loaded on the surface of UCNPs via the reactive oxygen species (ROSs) sensitive chemical bond. After the transfection and incubation, both of the upconversion nanoprobe and the genetically-encoded photosensitizer were accumulated in the mitochondria of cancer cells. Under the NIR irradiation, the emission of UCNPs could excite the expressed protein photosensitizer to generate ROSs which then stimulated the release of siRNAs in a controllable manner, achieving PDT and cascade gene therapy. Since the generation of ROSs and the release of siRNA occurred in the mitochondria in-situ, the mitochondria-mediated cell apoptosis signal pathway would be activated to induce cell apoptosis and subsequently inhibit tumor growth. To the best of our knowledge, this is the first report about NIR laser-activated, organelle-localized genetically-encoded photosensitizers developed for cascade therapy, which will widen the application of optogenetic tools in the tumor therapy. STATEMENT OF SIGNIFICANCE: The application of genetically-encoded photosensitizers in photodynamic therapy (PDT) is mainly limited by the shallow tissue penetration depth of the excitation light and unsatisfactory therapeutic performance. In this experiment, we developed an upconversion nanoparticles-based optogenetic nanosystem to enhance the PDT and cascade gene therapy for malignant tumors. The expressed genetically-encoded photosensitizers were accumulated in the mitochondria, which were activated in situ by the upconversion nanoprobe. Besides, the photogenerated reactive oxygen species (ROSs) stimulated the release of siRNAs in a controllable manner. To the best of our knowledge, this is the first report about NIR laser-activated, genetically-encoded photosensitizers developed for organelle-localized controllable cascade therapy. We hope this work can accelerate the application of genetically-encoded photosensitizers in the tumor therapy.

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Acoustic-based imaging techniques, including ultrasonography and photoacoustic imaging, are powerful noninvasive approaches for tumor imaging owing to sound transmission facilitation, deep tissue penetration, and high spatiotemporal resolution. Usually, imaging modes were classified into "always-on" mode and "activatable" mode. Conventional "always-on" acoustic-based probes often have difficulty distinguishing lesion regions of interest from surrounding healthy tissues due to poor target-to-background signal ratios. As compared, activatable probes have attracted attention with improved sensitivity, which can boost or amplify imaging signals only in response to specific biomolecular recognition or interactions. The tumor microenvironment (TME) exhibits abnormal physiological conditions that can be used to identify tumor sections from normal tissues. Various types of organic dyes and biomaterials can react with TME, leading to obvious changes in their optical properties. The TME also affects the self-assembly or aggregation state of nanoparticles, which can be used to design activatable imaging probes. Moreover, acoustic-based imaging probes and therapeutic agents can be coencapsulated into one nanocarrier to develop nanotheranostic probes, achieving tumor imaging and cooperative therapy. Satisfactorily, ultrasound waves not only accelerate the release of encapsulated therapeutic agents but also activate therapeutic agents to exert or enhance their therapeutic performance. Meanwhile, various photoacoustic probes can convert photon energy into heat under irradiation, achieving photoacoustic imaging and cooperative photothermal therapy. In this review, we focus on the recently developed TME-triggered ultrasound and photoacoustic theranostic probes for precise tumor imaging and targeted tumor therapy.

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A sequentially responsive nanocarrier was fabricated with three-in-one functional integration: bio-imaging, tumor microenvironment responsive chemotherapy and cascade activation of upconversion photodynamic therapy. The designed DNA outer nanoshell displayed site-specific degradation and controlled degradation speed. Significantly, the developed controllable nanotheranostic agent displayed high cell apoptosis ratios and obvious tumor inhibition.

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Objective To investigate the relationship between inflammatory factors, oxidative stress and type 1 deiodinase (DIO-1) concentration in patients with chronic renal failure (CRF) with or without euthyroid sick syndrome (ESS). Methods This study recruited patients with CRF and divided them into two groups: group 1 had low free triiodothyronine (FT3) levels; and group 2 had normal FT3 levels. Group 3 consisted of healthy volunteers. Serum levels of interleukin (IL)-6, IL-1ß, tumour necrosis factor (TNF)-α, 8-isoprostane and DIO-1 were measured using enzyme-linked immunosorbent assays. Multiple regression analysis was used to analyse correlations between parameters. Results Sixty patients were enrolled into each group and the groups were comparable in terms of vital signs, white blood cell count, free thyroxine and thyroid stimulating hormone concentrations. The serum DIO-1 concentration was significantly higher in group 2 than in groups 1 and 3. Multivariate regression analysis revealed that the DIO-1 concentration was inversely correlated with the TNF-α concentration. Conclusions Patients with CRF without ESS showed higher concentrations of DIO-1 than patients with ESS. The DIO-1 concentration was inversely correlated with the TNF-α concentration, which might indicate that the inflammatory response was milder in the patients with CRF without ESS than in those with ESS.

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