High-Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super-Resolution Imaging.

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4-9 frame binning, we achieved a 2-3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

Multimode Time-Resolved Superresolution Microscopy Revealing Chain Packing and Anisotropic Single Carrier Transport in Conjugated Polymer Nanowires.

Here, we developed a novel, multimode superresolution method to perform full-scale structural mapping and measure the energy landscape for single carrier transport along conjugated polymer nanowires. Through quenching of the local emission, the motion of a single photogenerated hole was tracked using blinking-assisted localization microscopy. Then, utilizing binding and unbinding dynamics of quenchers onto the nanowires, local emission spectra were collected sequentially and assembled to create a superresolution map of emission sites throughout the structure. The hole polaron trajectories were overlaid with the superresolution maps to correlate structures with charge transport properties. Using this method, we compared the efficiency of inter- and intrachain hole transport inside the nanowires and for the first time directly measured the depth of carrier traps originated from torsional disorder and chemical defects.

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.

High-Throughput Counting and Superresolution Mapping of Tetraspanins on Exosomes Using a Single-Molecule Sensitive Flow Technique and Transistor-like Semiconducting Polymer Dots.

A method for high-throughput counting and superresolution mapping of surface proteins on exosomes is described. The method combines a single-molecule sensitive flow technique and an adaptive superresolution imaging method. Exosomes stained with membrane dye and dye-conjugated antibodies were analyzed using a microfluidic platform at a flow rate of 100 exosome s-1 to determine size and protein copy number. Superresolution mapping was performed with exosomes labeled with novel transistor-like, semiconducting polymer dots (Pdots), which exhibit spontaneous blinking with <5 nm localization error and a broad range of optical-adjustable duty cycles. Based on the copy numbers extracted from the flow analysis, the switch-on frequency of the Pdots were finely adjusted so that structures of hundreds of exosomes were obtained within five minutes. The high throughput and high sensitivity of this method offer clear advantages for characterization of exosomes and similar biological vesicles.

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.

Dual-Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots.

In a conjugated polymer-based single-particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single-particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin-coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.

Compact Conjugated Polymer Dots with Covalently Incorporated Metalloporphyrins for Hypoxia Bioimaging.

Hypoxia is closely related to multiple diseases, especially in tumors, which increases the aggressiveness and drug resistance of cancer cells. Precise hypoxia imaging is of great significance for cancer diagnosis and the evaluation of therapeutic effects. A kind of hydrophobic polymer (i.e., PFPtTFPP) as an imaging probe for hypoxia with fluorene as an energy donor and an oxygen-sensitive PtII porphyrin as an energy acceptor was developed. Compact polymer dots (Pdots) with a small size were prepared by nanoprecipitation. The PFPtTFPP Pdots showed excellent hypoxia sensing in solution with high sensitivity and full reversibility. The emission intensity, quantum yields, lifetime, and single-particle brightness significantly increased under hypoxia conditions. Remarkably, hypoxia imaging in vitro and in vivo was realized, and a clear increase in brightness was observed under hypoxia conditions and in the tumor area. Excellent hypoxia imaging ability is beneficial to potential applications in cancer diagnosis.

Semiconducting Polymer Nanocavities: Porogenic Synthesis, Tunable Host-Guest Interactions, and Enhanced Drug/siRNA Delivery.

Nanocavities composed of lipids and block polymers have demonstrated great potential in biomedical applications such as sensors, nanoreactors, and delivery vectors. However, it remains a great challenge to produce nanocavities from fluorescent semiconducting polymers owing to their hydrophobic rigid polymer backbones. Here, we describe a facile, yet general strategy that combines photocrosslinking with nanophase separation to fabricate multicolor, water-dispersible semiconducting polymer nanocavities (PNCs). A photocrosslinkable semiconducting polymer is blended with a porogen such as degradable macromolecule to form compact polymer dots (Pdots). After crosslinking the polymer and removing the porogen, this approach yields semiconducting polymer nanospheres with open cavities that are tunable in diameter. Both small molecules and macromolecules can be loaded in the nanocavities, where molecular size can be differentiated by the efficiency of the energy transfer from host polymer to guest molecules. An anticancer drug doxorubicin (Dox) is loaded into the nanocavities and the intracellular release is monitored in real time by the fluorescence signal. Finally, the efficient delivery of small interfering RNA (siRNA) to silence gene expression without affecting cell viability is demonstrated. The combined features of bright fluorescence, tunable cavity, and efficient drug/siRNA delivery makes these nanostructures promising for biomedical imaging and drug delivery.

Therapeutic Considerations and Conjugated Polymer-Based Photosensitizers for Photodynamic Therapy.

Conjugated polymers have recently attracted a great deal of attention for applications in photodynamic therapy (PDT) because of their light-harvesting capability, efficient energy transfer, and singlet oxygen generation properties. This review describes recent advances in PDT development, including therapeutic mechanisms of PDT in cancer treatments, light excitation methods, and especially recent advances of conjugated polyelectrolytes and conjugated polymer nanoparticles as photosensitizers. The future direction on PDT and further development of conjugated polymer photosensitizers are discussed. The aim of this review is to stimulate innovative ideas to synthesize a new generation of conjugated polymer photosensitizers and promote their translation to clinical applications of PDT.

OCT imaging detection of brain blood vessels in mouse, based on semiconducting polymer nanoparticles.

Optical Coherence Tomography (OCT) is a valuable technology that has been used to obtain microstructure images of tissue, and has several advantages, though its applications are limited in high-scattering tissues. Therefore, semiconducting polymer nanoparticles (SPNs) that possess strong absorption characteristics are applied to decrease light scattering in tissues and used as exogenous contrast agents for enhancing the contrast of OCT imaging detection. In this paper, we prepared two kinds of SPNs, termed PIDT-TBZ SPNs and PBDT-TBZ SPNs, as the contrast agents for OCT detection to enhance the signal. Firstly, we proved that they were good contrast agents for OCT imaging in agar-TiO2. After that, the contrast effects of these two SPNs were quantitatively analyzed, and then cerebral blood vessels were monitored by a home-made SD-OCT system. Finally, we created OCT images in vitro and in vivo with these two probes and performed quantitative analysis using the images. The results indicated that these SPNs created a clear contrast enhancement of small vessels in the OCT imaging process, which provides a basis for the application of SPNs as contrast agents for bioimaging studies.

Semiconducting Polymer Dots for Point-of-Care Biosensing and In Vivo Bioimaging: A Concise Review.

In recent years, semiconducting polymer dots (Pdots) have attracted much attention due to their excellent photophysical properties and applicability, such as large absorption cross section, high brightness, tunable fluorescence emission, excellent photostability, good biocompatibility, facile modification and regulation. Therefore, Pdots have been widely used in various types of sensing and imaging in biological medicine. More importantly, the recent development of Pdots for point-of-care biosensing and in vivo imaging has emerged as a promising class of optical diagnostic technologies for clinical applications. In this review, we briefly outline strategies for the preparation and modification of Pdots and summarize the recent progress in the development of Pdots-based optical probes for analytical detection and biomedical imaging. Finally, challenges and future developments of Pdots for biomedical applications are given.

The effect of insulin and kruppel like factor 10 on osteoblasts in the dental implant osseointegration in diabetes mellitus patients.

Diabetes mellitus, metabolic disease, is characterized by chronic hyperglycemia. Patients with diabetes mellitus are susceptible to infection and therefore have a higher prevalence and progression rate of periodontal disease. We aimed to study the effect of insulin and kruppel like factor 10 (KLF10) on osteoblasts proliferation and differentiation, and expression of bone metabolism-related molecules and related signaling pathway molecules of AKT serine/threonine kinase 1 (AKT) and nuclear factor kappa B subunit 1 (NF-κB) through in vitro experiments, which can provide theoretical basis for the dental implant osseointegration in diabetic patients. The osteoblasts (hFOB 1.19 cells) were subdivided into KLF10 gene over expression group, KLF10 gene knockdown group, and KLF10 gene knockdown + insulin treatment group. CCK-8 and ELISA were, respectively, used for analysis of cell proliferation and differentiation. In vitro experiments were applied to detect the mRNA and protein expression of bone metabolism-related molecules, respectively. GSE178351 dataset and GSE156993 dataset were utilized to explore the expression of KLF10 in periodontitis. In osteoblasts, insulin treatment increased the expression of KLF10. Insulin and KLF10 could reduce the proliferation and differentiation of osteoblasts. Knockdown of KLF10 could increase the expression of bone metabolism-related molecules and activate AKT and NF-κB pathways, whereas insulin reversed this effect. KLF10 was up-regulated in both patients with periodontitis and type 2 diabetes mellitus with periodontitis. It is assumed that knockdown of KLF10 in insulin resistance may promote osteoblasts differentiation and dental implant osseointegration in diabetic patients.

Enhancing the Long-Term Stability of a Polymer Dot Glucose Transducer by Using an Enzymatic Cascade Reaction System.

Impaired glucose metabolism in diabetes causes severe acute and long-term complications, making real-time detection of blood glucose indispensable for diabetic patients. Existing continuous glucose monitoring systems are unsuitable for long-term clinical glycemic management due to poor long-term stability. Polymer dot (Pdot) glucose transducers are implantable optical nanosensors that exhibit excellent brightness, sensitivity, selectivity, and biocompatibility. Here, it is shown that hydrogen peroxide-a product of glucose oxidation in Pdot glucose sensors-degrades sensor performance via photobleaching, reduces glucose oxidase activity, and generates cytotoxicity. By adding catalase to a glucose oxidase-based Pdot sensor to create an enzymatic cascade, the hydrogen peroxide product of glucose oxidation is rapidly decomposed by catalase, preventing its accumulation and improving the sensor's photostability, enzymatic activity, and biocompatibility. Thus, a next-generation Pdot glucose transducer with a multienzyme reaction system (Pdot-GOx/CAT) that provides excellent sensing characteristics as well as greater detection system stability is presented. Pdot glucose transducers that incorporate this enzymatic cascade to eliminate hydrogen peroxide will possess greater long-term stability for improved continuous glucose monitoring in diabetic patients.

Second near-infrared photoactivatable biocompatible polymer nanoparticles for effective in vitro and in vivo cancer theranostics.

Photoacoustic imaging (PAI)-guided photothermal therapy (PTT) has drawn considerable attention due to the deeper tissue penetration and higher maximum permissible exposure. However, current phototheranostic agents are greatly restricted by weak absorption in the second near-infrared (NIR-II, 1000-1700 nm) window, long-term toxicity, and poor photostability. In this report, novel organic NIR-II conjugated polymer nanoparticles (CPNs) based on narrow bandgap donor-acceptor BDT-TBZ polymers were developed for effective cancer PAI and PTT. Characterization data confirmed the high photothermal conversion efficiency, good photostability, excellent PAI performance, and superior biocompatibility of as-obtained CPNs. In addition, in vitro and in vivo tests demonstrated the efficient PTT effect of CPNs in ablating cancer cells and inhibiting tumor growth under 1064 nm laser irradiation. More importantly, the CPNs exhibited rapid clearance capability through the biliary pathway and negligible systematic toxicity. Thus, this work provides a novel organic theranostic nanoplatform for NIR-II PAI-guided PTT, which advances the future clinical translation of biocompatible and metabolizable conjugated nanomaterials in cancer diagnosis and therapy.

Thermosensitive Polymer Dot Nanocomposites for Trimodal Computed Tomography/Photoacoustic/Fluorescence Imaging-Guided Synergistic Chemo-Photothermal Therapy.

Precision delivery of theranostic agents to the tumor site is essential to improve their diagnostic and therapeutic efficacy and concurrently minimize adverse effects during treatment. In this study, a novel concept of near-infrared (NIR) light activation of conjugated polymer dots (Pdots) at thermosensitive hydrogel nanostructures is introduced for multimodal imaging-guided synergistic chemo-photothermal therapy. Interestingly, owing to the attractive photothermal conversion efficiency of Pdots, the Pdots@hydrogel as theranostic agents is able to undergo a controllable softening or melting state under the irradiation of NIR laser, resulting in light-triggered drug release in a controlled way and concurrently hydrogel degradation. Besides, the novel Pdots@hydrogel nanoplatform can serve as the theranostic agent for enhanced trimodal photoacoustic (PA)/computed tomography (CT)/fluorescence (FL) imaging-guided synergistic chemo-photothermal therapy of tumors. More importantly, the constructed intelligent nanocomposite Pdots@hydrogel exhibits excellent biodegradability, strong NIR absorption, bright PA/CT/FL signals, and superior tumor ablation effect. Therefore, the concept of a light-controlled multifunctional Pdots@hydrogel that integrates multiple diagnostic/therapeutic modalities into one nanoplatform can potentially be applied as a smart nanotheranostic agent to various perspectives of personalized nanomedicine.

Brightness Enhancement of Near-Infrared Semiconducting Polymer Dots for in Vivo Whole-Body Cell Tracking in Deep Organs.

In vivo visualization of cell migration and engraftment in small animals provide crucial information in biomedical studies. Semiconducting polymer dots (Pdots) are emerging as superior probes for biological imaging. However, in vivo whole-body fluorescence imaging is largely constrained by the limited brightness of Pdots in near-infrared (NIR) region. Here, we describe the brightness enhancement of NIR fluorescent Pdots for in vivo whole-body cell tracking in deep organs. We first synthesize semiconducting polymers with strong absorption in orange and far-red regions. By molecular doping, the weak broad-band fluorescence of the Pdots was significantly narrowed and enhanced by 1 order of magnitude enhancement, yielding bright narrow-band NIR emission with a quantum yield of ∼0.21. Under an excitation of far-red light (676 nm), a trace amount of Pdots (∼2 µg) in the stomach can be clearly detected in whole-body fluorescence imaging of live mice. The Pdots coated with a cell-penetrating peptide are able to brightly label cancer cells with minimal cytotoxicity. In vivo cell tracking in live mice indicated that the entrapment and migration of the tail-vein-administered cells (∼400 000) were clearly visualized in real time. These Pdots with deep-red excitation and bright NIR emission are promising for in vivo whole-body fluorescence imaging.

Photo-Cross-Linkable Polymer Dots with Stable Sensitizer Loading and Amplified Singlet Oxygen Generation for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising treatment modality for clinical cancer therapy. However, the therapeutic effect of PDT is strongly dependent on the property of photosensitizer. Here, we developed photo-cross-linkable semiconductor polymer dots doped with photosensitizer Chlorin e6 (Ce6) to construct a nanoparticle platform for photodynamic therapy. Photoreactive oxetane groups were attached to the side chains of the semiconductor polymer. After photo-cross-linking reaction, the Ce6-doped Pdots formed an interpenetrated structure to prevent Ce6 leaching out from the Pdot matrix. Spectroscopic characterizations revealed an efficient energy transfer from the polymer to Ce6 molecules, resulting in amplified generation of singlet oxygen. We evaluated the cellular uptake, cytotoxicity, and photodynamic effect of the Pdots in gastric adenocarcinoma cells. In vitro photodynamic experiments indicated that the Ce6-doped Pdots (∼10 µg/mL) effectively killed the cancer cells under low dose of light irradiation (∼60 J/cm2). Furthermore, in vivo photodynamic experiments were carried out in tumor-bearing nude mice, which indicated that the Pdot photosensitizer apparently suppressed the growth of solid tumors. Our results demonstrate that the photo-cross-linkable Pdots doped with photosensitizer are promising for photodynamic cancer treatment.

Real-Time Imaging of Endocytosis and Intracellular Trafficking of Semiconducting Polymer Dots.

Semiconducting polymer dots (Pdots) have shown great promise in biomedical applications, including biosensing, drug delivery, and live imaging of cells and biomolecules. Insight into the mechanism and regulation of cellular uptake and intracellular metabolism of Pdots is important for the development of superior Pdots-based theranostic nanoconjugates. Herein, we performed real-time imaging of endocytosis and intracellular trafficking of a type of fluorescent Pdots that showed excellent biocompatibility in various types of cells. The endocytic routes and kinetics of Pdots were differently regulated in distinct cell types. Following endocytosis, Pdots were transported in vesicles along microtubule and destined for lysosomes. Furthermore, our results revealed exosome-mediated extracellular release of Pdots and have tracked the dynamic process at the single particle level. These results provide new insight into the design of more effective and selective imaging probes as well as drug carriers.

Size-dependent property and cell labeling of semiconducting polymer dots.

Semiconducting polymer dots (Pdots) represent a new class of fluorescent nanoparticles for biological applications. In this study, we investigated their size-dependent fluorescence and cellular labeling properties. We demonstrate that the polymer conformation in solution phase largely affects the polymer folding and packing during the nanoparticle preparation process, resulting in solution-phase control over the fluorescence properties of semiconducting polymer nanoparticles. The resulting Pdots exhibit apparent size dependent absorption and emission, a characteristic feature of different chain packing behaviors due to the preparation conditions. Single-particle fluorescence imaging was employed to perform a side-by-side comparison on the Pdot brightness, indicating a quadratic dependence of single-particle brightness on particle size. Upon introducing a positively charged dye Nile blue, all the three type of Pdots were quenched very efficiently (Ksv > 1 × 10(7) M(-1)) in an applied quenching process at low dye concentrations, but exhibit apparent difference in quenching efficiency with increasing dye concentration. Furthermore, Pdots of different sizes were used for cell uptake and cellular labeling involving biotin-streptavidin interactions. Fluorescence imaging together with flow cytometry studies clearly showed size dependent labeling brightness. Small-sized Pdots appear to be more effective for immunolabeling of cell surface, whereas medium-sized Pdots exhibit the highest uptake efficiency. This study provides a concrete guidance for selecting appropriate particle size for biological imaging and sensing applications.