A Target-Triggered Emission Enhancement Strategy Based on a Y-Shaped DNA Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristic for microRNA Imaging in Living Cells.

DNA self-assembled fluorescent nanoprobes have been developed for bio-imaging owing to their high resistance to enzyme degradation and great cellular uptake capacity. In this work, we designed a new Y-shaped DNA fluorescent nanoprobe (YFNP) with aggregation-induced emission (AIE) characteristic for microRNA imaging in living cells. With the modification of the AIE dye, the constructed YFNP had a relatively low background fluorescence. However, the YFNP could emit a strong fluorescence due to the generation of microRNA-triggered AIE effect in the presence of target microRNA. Based on the proposed target-triggered emission enhancement strategy, microRNA-21 was detected sensitively and specifically with a detection limit of 122.8 pM. The designed YFNP showed higher bio-stability and cell uptake than the single-stranded DNA fluorescent probe, which has been successfully applied for microRNA imaging in living cells. More importantly, the microRNA-triggered dendrimer structure could be formed after the recognition of target microRNA, achieving a reliable microRNA imaging with a high spatiotemporal resolution. We expect that the proposed YFNP will become a promising candidate for bio-sensing and bio-imaging.

Long-Term In Vivo Glucose Monitoring by Polymer-Dot Transducer in an Injectable Hydrogel Implant.

Optical sensors have attracted a great deal of interest for glucose detection. However, their practical applications for continuous glucose monitoring are still constrained by operational reliability in subcutaneous tissues. Here, we show an implantable hydrogel platform embedded with luminescent polymer dots (Pdots) for sensitive and long-term glucose monitoring. We use Pdot transducer in a polyacrylamide hydrogel matrix to construct an implantable platform. The hydrogel-Pdot transducer showed bright luminescence with ratiometric response to glucose changes. The in vitro and in vivo sensitivities of the hydrogel implant were enhanced by varying the enzyme concentration and injection volume. After implantation, the hydrogel with Pdot transducer remained at the implanted site without migration for 1 month and can be removed from the subcutaneous tissue for further analysis. Our results indicate that the hydrogel-Pdot platform maintains the intrinsic sensing property with excellent stability during 1 month implantation, while fibrous capsule formation on the implant in some cases needs to be solved for long-term continuous glucose monitoring.

Near-Infrared Optical Transducer for Dynamic Imaging of Cerebrospinal Fluid Glucose in Brain Tumor.

Aberrant cerebral glucose metabolism is related to many brain diseases, especially brain tumor. However, it remains challenging to measure the dynamic changes in cerebral glucose. Here, we developed a near-infrared (NIR) optical transducer to sensitively monitor the glucose variations in cerebrospinal fluid in vivo. The transducer consists of an oxygen-sensitive nanoparticle combined with glucose oxidase (GOx), yielding highly sensitive NIR phosphorescence in response to blood glucose change. We demonstrated long-term continuous glucose monitoring by using the NIR transducer. After subcutaneous implantation, the glucose transducer provides a strong luminescence signal that can continuously monitor blood glucose fluctuations for weeks. By using the NIR emission of the transducer, we further observed abnormal dynamic changes in cerebrospinal fluid glucose and quantitatively assessed cerebral glucose uptake rates in transgenic mice bearing brain tumors. This study provides a promising method for the diagnosis of various metabolic diseases with altered glucose metabolism.

Improving the Accuracy of Pdot-Based Continuous Glucose Monitoring by Using External Ratiometric Calibration.

Continuous glucose monitoring (CGM) allows type I and II diabetes patients to track changes in their glucose levels, allowing detection of impending hypoglycemia or hyperglycemia. Polymer dots (Pdots) are candidates for use in implanted CGM systems due to their exceptional brightness, photostability, sensitivity, and biocompatibility. However, Pdot glucose transducers are oxygen-dependent, and changes in tissue oxygen levels affect their measurement accuracy. Here, we describe an external ratiometric calibration method that corrects for changes in tissue oxygen levels to improve measurement accuracy. This method uses the ratio of oxygen concentrations inside and outside the Pdot glucose transducer as an indicator of glucose concentration to correct for signal deviations caused by tissue oxygen fluctuations. A second oxygen-sensitive Pdot that is not conjugated with glucose oxidase is used to measure the oxygen concentration outside the Pdot glucose transducer. We describe the theoretical basis for this approach and demonstrate its effectiveness experimentally in a subcutaneous mouse implant model. This external ratiometric system achieves higher accuracy glucose measurements than previous Pdot-based CGM systems and comparable accuracy to current commercial CGM systems, demonstrating the utility of the external ratiometric calibration strategy.

Metabolic study of ginsenoside Rg3 and glimepiride in type 2 diabetic rats by liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry.

RATIONALE: Ginsenoside Rg3 and glimepiride have been applied to treat type 2 diabetes (T2DM) because of their good hypoglycemic effects. In this study, the effects of ginsenoside Rg3 acting synergistically with glimepiride were investigated in liver microsomes from rats with type 2 diabetes. METHODS: An in vitro incubation system with normal rat liver microsomes (RLM) and type 2 diabetic rat liver microsomes (TRLM) was developed. The system also included two experimental groups consisting of RLM and TRLM pretreated with ginsenoside Rg3 and glimepiride (named the RLMR and TRLMR groups, respectively). The metabolism in the different groups was analyzed by ultra-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (UPLC/Q-Orbitrap MS). RESULTS: The results showed that the concentration of glimepiride increased in RLM and TRLM after treatment with ginsenoside Rg3. Five metabolites (M1-M5) of glimepiride were found, and they were named 3N-hydroxyglimepiride, hydroxyglimepiride, 1,2-epoxy ether-3-hydroxyglimepiride, 1N-hydroxyglimepiride and 1N,2C,S,O,O-epoxy ether-3-hydroxyglimepiride. The metabolite of ginsenoside Rg3 was ginsenoside Rh2. CONCLUSIONS: An in vitro incubation system with RLM and TRLM was developed. The system revealed pathways that produce glimepiride metabolites. Ginsenoside Rg3 may inhibit the activity of cytochrome P450 enzymes in vitro. The present study showed that ginsenoside Rg3 and glimepiride may be combined for the treatment of T2DM.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age-related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point-of-care NADH sensing. Here we develop a series of NADH-sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene-based Pdots, this mechanism resulted in an on-off NADH sensor; in DPA-CNPPV Pdots, UV excitation resulted in NADH-sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA-CNPPV Pdots exhibits high sensitivity (3.1 µM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH-sensing Pdots including smartphone-based NADH imaging for point-of-care use.

Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-Specific Enzyme.

We introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and ß-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use.

Exosomal MALAT1 derived from ox-LDL-treated endothelial cells induce neutrophil extracellular traps to aggravate atherosclerosis.

The objective of this study was to reveal a novel mechanism underlying the progression of atherosclerosis (AS) associated with endothelial cells (ECs) and neutrophils. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology and particle size of isolated exosomes. Western blotting was applied to examine exosomal markers, while the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The production of inflammatory cytokines and reactive oxygen species (ROS) was determined by an enzyme-linked immunosorbent assay (ELISA) and a dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Circulating neutrophil extracellular traps (NETs) were represented by myeloperoxidase (MPO)-DNA complexes. NETs formation was assessed using immunofluorescence microscopy. Atherosclerotic lesion development was measured by Oil Red O (ORO) staining. In the results, MALAT1 expression was increased in exosomes extracted from oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). When co-cultured with human neutrophils, exosomes derived from ox-LDL-treated HUVECs were revealed to promote NETs formation, which was mediated by exosomal MALAT1. Furthermore, ox-LDL-treated HUVECs-derived exosomes were demonstrated to trigger hyperlipidemia, inflammatory response and NETs release in a mouse model of AS. In conclusion, exosomal MALAT1 derived from ox-LDL-treated ECs initiated NETs formation, which in turn deteriorated AS.

YY1-mediated PTEN dephosphorylation antagonizes IR-induced DNA repair contributing to tongue squamous cell carcinoma radiosensitization.

Ionizing radiation (IR) confers a survival advantage in tongue squamous cell carcinoma (TSCC), however, IR resistance limits its efficacy. Although Yin Yang 1 (YY1) has been reported to play a role in genotoxic drug resistance by accelerating DNA repair, its role in TSCC radioresistance remains unclear. In this study, we examined YY1 mRNA and protein expression in human tongue cancer samples using qRT-PCR and western blotting, respectively. DNA array data identified YY1 mRNA expression in IR sensitivity or resistance cell lines and tissues. Tongue carcinoma primary cells and CAL27 cells with YY1 stably overexpressed or knocked-down were exposed to IR and evaluated for cell proliferation and apoptosis by CCK8-assay and caspase-3 assay, respectively. We also examined DNA damage- or repair-related indicators, such as YY1, p-H2AX, nuclear PTEN, p-PTEN, and Rad51 through Western blot analysis. Additionally, we explored the mechanism of IR-induced PTEN nuclear translocation by introducing a series of PTEN phosphorylation site mutations and co-IP assay. We observed that YY1 mRNA and protein are highly expressed in TSCC tissues, which was correlated with worse overall survival. Moreover, higher expression of YY1 and Rad51 was observed in radioresistant cells and tissues, overexpression of YY1 led to IR resistance in TSCC cells, whereas YY1 knockdown sensitized TSCC cells to IR. The underlying mechanism showed that the overexpression of YY1 upregulated nuclear PTEN and Rad51 expression, which is essential for DNA repair. IR upregulated YY1, nuclear PTEN, and Rad51; thus, knockdown of YY1 completely blocked IR-induced upregulation of nuclear PTEN/Rad51. IR upregulated PTEN phosphorylation, and mutation of the phosphorylation site of Ser380 nearly completely blocked IR-induced PTEN nuclear translocation. Furthermore, the phosphatase PP2A negatively regulated pS380-PTEN, and knockdown of YY1 completely blocked IR-induced pS380-PTEN through PP2A. In conclusion, knockdown of YY1 enhanced TSCC radiosensitivity through PP2A-mediated dephosphorylation of PTEN Ser380; thus, antagonizing the IR-induced nuclear PTEN/Rad51 axis and targeting YY1 may reverse IR resistance in TSCC.

Platelet-neutrophil interaction aggravates vascular inflammation and promotes the progression of atherosclerosis by activating the TLR4/NF-κB pathway.

Platelet-neutrophil interaction is well known for its role in inflammatory diseases; however, its biological role in atherosclerosis (AS) progression remains unclear. Human peripheral blood neutrophils were obtained to compare toll-like receptor 4 (TLR4), tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and myeloid-related proteins 8/14 (Mrp8/14) levels in 22 AS patients with those in 18 healthy controls using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Meanwhile, mouse marrow neutrophils subjected to different treatment were collected for the ELISA assay, cell apoptosis, and Western blot analysis. Normal diet or high-fat diet ApoE-/- mice with or without administration of Mrp8/14 antagonist paquinimod were used for plasma collection to measure total cholesterol, triglycerides, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, TNF-α, IL-1ß, Mrp8/14, TLR4, and nuclear factor (NF)-κB p65 levels. The results showed that Mrp8/14 and TLR4-mediated inflammatory pathway was activated in neutrophils of AS patients. In vitro experiments demonstrated that platelet-neutrophil interaction promoted the Mrp8/14 release and inhibited neutrophil apoptosis via P-selectin. Furthermore, platelet-neutrophil interaction upregulated TLR4/myeloid differentiation factor 88/NF-κB pathway. Conversely, Mrp8/14/TLR4/NF-κB interference alleviated AS progression. In conclusion, Mrp8/14/TLR4/NF-κB activated by platelet-neutrophil interaction is an important inflammatory signaling pathway for AS pathogenesis.

A BODIPY-Based Donor/Donor-Acceptor System: Towards Highly Efficient Long-Wavelength-Excitable Near-IR Polymer Dots with Narrow and Strong Absorption Features.

Bright long-wavelength-excitable semiconducting polymer dots (LWE-Pdots) are highly desirable for in vivo imaging and multiplexed in vitro bioassays. LWE-Pdots have been obtained by incorporating a near-infrared (NIR) emitter into the backbone of a polymer host to develop a binary donor-acceptor (D-A) system. However, they usually suffer from severe concentration quenching and a trade-off between fluorescence quantum yield (Φf ) and absorption cross-section (σ). Herein, we describe a ternary component (D1 /D2 -A) strategy to achieve ultrabright, green laser-excitable Pdots with narrow-band NIR emission by introducing a BODIPY-based assistant polymer donor as D1 . The D1 /D2 -A Pdots possess improved Φf and σ compared to corresponding binary D2 -A Pdots. Their Φf is as high as 40.2 %, one of the most efficient NIR Pdots reported. The D1 /D2 -A Pdots show ultrahigh single-particle brightness, 83-fold brighter than Qdot 705 when excited by a 532 nm laser. When injected into mice, higher contrast in vivo tumor imaging was achieved using the ternary Pdots versus the binary D-A Pdots.

Purification of Semiconducting Polymer Dots by Size Exclusion Chromatography Prior to Cytotoxicity Assay and Stem Cell Labeling.

Semiconducting polymer dots (Pdots) as fluorescent probes have shown promising applications because of their excellent optical properties. However, apparent differences were observed in cytotoxicity assays, which might originate from impurities introduced in polymer synthesis or nanoparticle preparation. A simple gel-filtration-based purification method was used to address this issue. Purified Pdots displayed obviously decreased cytotoxicity as compared with the same batch of unpurified Pdots. The purified Pdots were further examined in a cytotoxicity study on mesenchymal stem cells (MSCs), which are very sensitive to exogenous probes. The results indicated that purified Pdots did not affect the proliferation ability of MSCs, while unpurified Pdots could have obvious cytotoxicity. In addition, the purified Pdots did not show cytotoxicity even after 6 months of storage. Our results demonstrated that gel filtration is an effective method for obtaining Pdots with minimal cytotoxicity, which are more suitable for biological applications.

Improving the Photostability of Semiconducting Polymer Dots Using Buffers.

The photostability of fluorescent probes is critical in biological imaging, especially for long-term observational analyses. Here, we describe a simple and universal method to improve the photostability of semiconducting polymer dots (Pdots) and other fluorescent probes by using buffers. Using Pdots as a model system, we found that HEPES or MES buffer can improve the photostability of Pdots by a factor of 20. Through a systematic study, we show that Pdot photobleaching is dominated by photoinduced radicals which can be quenched by the piperazine or morpholine structures of these buffers, which act as radical scavengers. For conditions where choice of buffer is limited, we designed fluorescent polymers conjugated with radical scavengers to improve Pdot photostability. We then demonstrate a practical application in which HEPES buffer is used to improve the photostability of Pdots during cell imaging.

Ratiometric Barcoding for Mass Cytometry.

Barcoding is of importance for high-throughput cellular and molecular analysis. A ratiometric barcoding strategy using lanthanide-coordinated polymer dots (Ln-Pdots) was developed for mass cytometric analysis. By using 3 metal isotopes and 4 ratio intensity levels, 16 barcodes were generated to code, and later decode, cell samples in mass cytometry. The ratiometric Ln-Pdot barcodes not only provided high-mass-signal intensities but also eliminated the bias caused by different concentrations of the labeling reagents/barcodes and run-to-run differences in cell labeling efficiency. The ability to distinguish clearly the 16 sets of labeled MCF-7 cells with mass cytometry demonstrated the excellent resolving power of the ratiometric Ln-Pdot barcodes. Furthermore, the results from barcoding PBMC samples via CD45-specific cellular targeting indicated that the ratiometric Ln-Pdot barcodes could facilitate mass cytometry in high-throughput and multiplexed analysis, especially with precious human samples.

Single-Molecule Flow Platform for the Quantification of Biomolecules Attached to Single Nanoparticles.

We describe here a flow platform for quantifying the number of biomolecules on individual fluorescent nanoparticles. The platform combines line-confocal fluorescence detection with near nanoscale channels (1-2 µm in width and height) to achieve high single-molecule detection sensitivity and throughput. The number of biomolecules present on each nanoparticle was determined by deconvolving the fluorescence intensity distribution of single-nanoparticle-biomolecule complexes with the intensity distribution of single biomolecules. We demonstrate this approach by quantifying the number of streptavidins on individual semiconducting polymer dots (Pdots); streptavidin was rendered fluorescent using biotin-Alexa647. This flow platform has high-throughput (hundreds to thousands of nanoparticles detected per second) and requires minute amounts of sample (∼5 µL at a dilute concentration of 10 pM). This measurement method is an additional tool for characterizing synthetic or biological nanoparticles.

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid.

Developing probes for the detection of reactive oxygen species (ROS), a hallmark of many pathophysiological process, is imperative to both understanding the precise roles of ROS in many life-threatening diseases and optimizing therapeutic interventions. We herein report an all-in-one fluorescent semiconducting polymer based far-red to near-infrared (NIR) Pdot nanoprobe for the ratiometric detection of hypochlorous acid (HOCl). The fabrication takes the advantage of flexible polymer design by incorporating target-sensitive and target-inert fluorophores into a single conjugated polymer to avoid leakage or differential photobleaching problems existed in other nanoprobes. The obtained nanoprobe has improved performance in HOCl sensing, such as high brightness, ideal far-red to NIR optical window, excellent photostability, self-referenced ratiometric response, fast response, and high selectivity. The dual-emission property allows the sensitive imaging of HOCl fluctuations produced in living macrophage cells and peritonitis of living mice with high contrast. This study not only provides a powerful and promising nanoprobe to be potentially used in the investigations of in situ HOCl status of diseases in living systems but also puts forward the design strategy of a new category of ratiometric fluorescent probes facilitating precise and reliable measurement in biological systems.

Optically Encoded Semiconducting Polymer Dots with Single-Wavelength Excitation for Barcoding and Tracking of Single Cells.

Multiplexed optical encoding is emerging as a powerful technique for high-throughput cellular analysis and molecular assays. Most of the developed optical barcodes, however, either suffer from large particle size or are incompatible with most commercial optical instruments. Here, a new type of nanoscale fluorescent barcode (Pdot barcodes) was prepared from semiconducting polymers. The Pdot barcodes possess the merits of small size (∼20 nm in diameter), narrow emission bands (full-width-at-half-maximum (fwhm) of 30-40 nm), three-color emissions (blue, green, and red) under single-wavelength excitation, a high brightness, good pH and thermal stability, and efficient cellular uptake. The Pdot barcodes were prepared using a three-color and six-intensity encoding strategy; for ratiometric readout of the barcodes, one of the colors might be used as an internal reference. We used the Pdot barcodes to label 20 sets of cancer cells and then distinguished and identified each set based on the Pdot barcodes using flow cytometry. We also monitored and tracked single cells labeled with different Pdot barcodes, even through rounds of cell division. These results suggest Pdot barcodes are strong candidates for discriminating different labeled cell and for long-term cell tracking.

Lanthanide-Coordinated Semiconducting Polymer Dots Used for Flow Cytometry and Mass Cytometry.

Simultaneous monitoring of biomarkers as well as single-cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling-network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide-coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual-functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.

Squaraine-based polymer dots with narrow, bright near-infrared fluorescence for biological applications.

This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.

Single-chain semiconducting polymer dots.

This work describes the preparation and validation of single-chain semiconducting polymer dots (sPdots), which were generated using a method based on surface immobilization, washing, and cleavage. The sPdots have an ultrasmall size of ∼3.0 nm as determined by atomic force microscopy, a size that is consistent with the anticipated diameter calculated from the molecular weight of the single-chain semiconducting polymer. sPdots should find use in biology and medicine as a new class of fluorescent probes. The FRET assay this work presents is a simple and rapid test to ensure methods developed for preparing sPdot indeed produced single-chain Pdots as designed.