Conjugated Polymer Nanoparticles for Cell Membrane Imaging

The outstanding optical properties and biocompatibility of fluorescent conjugated polymer nanoparticles (CPNs) make them favorable for bioimaging application. However, few CPNs could achieve stable cell membrane labeling due to cell endocytosis. In this work, conjugated polymer nanoparticles (PFPNP‐PLE) encapsulated with PFP and PLGA‐PEG‐N₃ in the matrix and functionalized with the small‐molecule drug plerixafor (PLE) on the surface were prepared by a mini‐emulsion method. PFPNP‐PLE exhibits excellent photophysical properties, low cytotoxicity, and specific cytomembrane location, which makes it a potential cell membrane labeling reagent with blue fluorescence emission, an important component for multilabel/multicolor bioimaging.     

Polyfluorene based conjugated polymer nanoparticles for two-photon live cell imaging

Two-photon excitation microscopy (2PEM) has been known as a noninvasive and powerful bio-imaging tool for studying living cells, intact tissues and living animals because of their unique advantages such as localized excitation, deep tissue penetration as well as less photo-damage. However, the major limitations that hinder its practical applications in biological systems are low two-photon absorption cross sections of conventional fluorescence probes. Conjugated polymer nanoparticles (CPNs) consisting of highly fluorescent conjugated polymers are promising fluorescent probes for 2PEM due to their unique advantages including large two-photon absorption cross sections, high fluorescence quantum yield, good photo-stability and biocompatibility, facile chemical synthesis, tunable optical properties as well as versatile surface modifications. This account summarizes the recent efforts of our group on development of novel polyfluorene based CPNs as 2PEM contrast agents for live cell imaging.     

Development of Photoactivated Fluorescent N‐Hydroxyoxindoles and Their Application for Cell‐Selective Imaging

Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N‐hydroxyoxindoles formed through the intramolecular photocyclization of substituted o‐nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra‐high fluorescence enhancement (up to 800‐fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure–activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE‐based fluorescent probes with desired photophysical and biological properties. As a proof‐of‐concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365–405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs).     

Photo‐ and pH‐Tunable Multicolor Fluorescent Nanoparticle‐Based Spiropyran‐ and BODIPY‐Conjugated Polymer with Graphene Oxide

We report a stimuli‐responsive fluorescent nanomaterial, based on graphene oxide coupled with a polymer conjugated with photochromic spiropyran (SP) dye and hydrophobic boron dipyrromethane (BODIPY) dye, for application in triggered target multicolor bioimaging. Graphene oxide (GO) was reduced by catechol‐conjugated polymers under mildly alkaline conditions, which enabled to formation of functionalized multicolor graphene nanoparticles that can be induced by irradiation with UV light and by changing the pH from acidic to neutral. Investigation of these nanoparticles by using AFM, fluorescence emission, and in vitro cell and in vivo imaging revealed that they show different tunable colors in bioimaging applications and, more specifically, in cancer‐cell detection. The stability, biocompatibility, and quenching efficacy of this nanocomposite open a different perspective for cell imaging in different independent colors, sequentially and simultaneously.     

Tracking Cancer Metastasis In Vivo by Using an Iridium‐Based Hypoxia‐Activated Optical Oxygen Nanosensor

We have developed a nanosensor for tracking cancer metastasis by noninvasive real‐time whole‐body optical imaging. The nanosensor is prepared by the formation of co‐micelles from a poly(N‐vinylpyrrolidone)‐conjugated iridium(III) complex (Ir‐PVP) and poly(ε‐caprolactone)‐b‐poly(N‐vinylpyrrolidone) (PCL‐PVP). The near‐infrared phosphorescence emission of the nanosensor could be selectively activated in the hypoxic microenvironment induced by cancer cells. The detection ability of the nanosensor was examined in cells and different animal models. After intravenous injection, the nanosensor can be effectively delivered to the lung and lymph node, and cancer cell metastasis through bloodstream or lymphatics can be quickly detected with high signal‐to‐background ratio by whole‐body imaging and organ imaging. Moreover, the nanosensor exhibits good biocompatibility both in vitro and in vivo. The nanosensor is believed to be a powerful tool for the diagnosis of cancer metastasis.     

Solar‐Powered Organic Semiconductor–Bacteria Biohybrids for CO2 Reduction into Acetic Acid

An organic semiconductor–bacteria biohybrid photosynthetic system is used to efficiently realize CO₂ reduction to produce acetic acid with the non‐photosynthetic bacteria Moorella thermoacetica. Perylene diimide derivative (PDI) and poly(fluorene‐co‐phenylene) (PFP) were coated on the bacteria surface as photosensitizers to form a p‐n heterojunction (PFP/PDI) layer, affording higher hole/electron separation efficiency. The π‐conjugated semiconductors possess excellent light‐harvesting ability and biocompatibility, and the cationic side chains of organic semiconductors could intercalate into cell membranes, ensuring efficient electron transfer to bacteria. Moorella thermoacetica can thus harvest photoexcited electrons from the PFP/PDI heterojunction, driving the Wood–Ljungdahl pathway to synthesize acetic acid from CO₂ under illumination. The efficiency of this organic biohybrid is about 1.6 %, which is comparable to those of reported inorganic biohybrid systems.     

Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress.     

Water‐soluble dendritic‐conjugated polyfluorenes: Synthesis,characterization, and interactions with DNA

Three generation of Boc‐protected dendritic‐conjugated polyfluorenes ( Boc‐PFP‐G_0‐2 ) were synthesized by Suzuki coupling 1,4‐phenyldiboronic ester with dendritic monomers that were synthesized through generation‐by‐generation approach. The gel permeation chromatography (GPC) analyses showed that the weight‐average molecular weight (M_w) of Boc‐PFP‐G_0‐2 was in the range of 11,400–20,400 Da with the polydispersity index (PDI) in the range of 1.32–1.96. Treatment of Boc‐protected polymers with 6 M HCl in dioxane yielded cationic dendritic‐conjugated polyfluorenes ( PFP‐G_0‐2 ). They were soluble in common polar solvents such as DMSO, DMF, and water with absorption maxima between 345 and 379 nm. The solutions of PFP‐G_0‐2 in water were highly fluorescent with emission maxima between 416 and 425 nm. Because higher generation dendrons could prevent the formation of π‐stacking aggregates of backbones of conjugated polymer, the fluorescence quantum efficiencies (QEs) of PFP‐G_0‐2 enhance as the dendritic generation grew. The interactions between 25 mer double‐stranded DNA (dsDNA) and PFP‐G_0‐2 were studied using ethidium bromide (EB) as fluorescent probe. The electrostatic bindings of PFP‐G_0‐2 with dsDNA/EB complex result in displacement of EB from DNA double helix to the solution accompanying by a quenching of EB fluorescence. The PFP‐G₂ with highest generation of dendritic side chains possessed a highest charge density and could form most stable complex with dsDNA. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7462–7472, 2008     

Intermolecular electronic coupling of 9-methyl-9H-dibenzo [a,c] carbazole for strong emission in aggregated state by substituent effect

Bright emission of organic luminogens at aggregated state has attracted increasing attention for their potential applications in opto-electronic devices and bio-/chemo-sensors. In this article, upon the introduction of different substituents(Br, Ph and TPh) to the large conjugated core of 9-methyl-9H-dibenzo[a,c]carbazole(DBC) moiety, the resultant luminogens demonstrated PL quantum yields in solid state ranging from 4.81% to 47.39%. Through the systematic investigation of molecular packing,together with theory calculation, the strong intermolecular electronic coupling in the dimers is proved as the main factor to the bright emission in the solid state. The results afforded a new avenue to investigate the intrinsic relationship among the molecular structures, packing modes and emission properties.     

环状RGDs修饰的分子刷型共聚物在成像及药物运输中的应用

以具有丰富接枝侧链的阴离子型共轭聚合物分子刷PFPANa为材料,通过简单的一步修饰法在聚合物的部分接枝侧链上引入靶向配体分子c(RGDyK),并利用分子刷侧链上未修饰配体分子的羧基负离子与抗癌药物DOX静电结合,制备了基于分子刷型共轭聚合物的靶向细胞成像和载药系统.研究结果表明载药系统对DOX药物的载药量可达13.3 wt%,体外细胞实验研究结果表明该载药系统可实现对肿瘤细胞的靶向选择性成像,并显著促进了肿瘤细胞对DOX药物的摄取,具有良好的抗肿瘤细胞生长效果,显著提高了药物运输效率.     

Highly efficient and non-doped red conjugated polymer dot for photostable cell imaging

By introducing a naphthothiadiazole (NT) unit as the main building block, a non-doped and red emissive conjugated polymer poly(9,9-dihexylfluorene-alt-naphthothiadiazole) (PFNT) is readily obtained through a two-step synthesis. Since the NT unit has a large twist angle with its neighboring segment, the aggregation-induced quenching (AIQ) effect of PFNT can be effectively suppressed in the condensed state. As a result, the corresponding PFNT polymer dot (Pdot) exhibits a high fluorescence quantum yield of 53.2% with peak emission at 616 nm, which is one of the most efficient red Pdots known. PFNT Pdot shows good biocompatibility and can be employed for living cell fluorescent imaging with high brightness. It also can be used for specific subcellular organelle imaging through immunofluorescence labeling. Furthermore, the PFNT Pdot demonstrates much better photostability for long-time cell fluorescence imaging than commercial red dyes. The high performances of PFNT Pdot make it a promising fluorescent probe for practical bioapplications.     

Large Two‐Photon Absorption of Terpyridine‐Based Quadrupolar Derivatives: Towards their Applications in Optical Limiting and Biological Imaging

Developing organic chromophores with large two‐photon absorption (TPA) in both organic solvents and aqueous media is crucial owing to their applications in solid‐state photonic devices and biological imaging. Herein, a series of novel terpyridine‐based quadrupolar derivatives have been synthesized. The influences of electron‐donating group, type of conjugated bridge, as well as solvent polarity on the molecular TPA properties have been investigated in detail. In contrast to the case in organic solvents, bis(thienyl)‐benzothiadiazole as a rigid conjugated bridge will completely quench molecular two‐photon emission in aqueous media. However, the combination of alkylcarbazole as the donor and bis(styryl)benzene as a conjugation bridge can enlarge molecular TPA cross‐sections in both organic solvent and aqueous media. The reasonable two‐photon emission brightness for the organic nanoparticles of chromophores 3 – 5 in the aqueous media, prepared by the reprecipitation method, enables them to be used as probes for in vivo biological imaging.     

Fluorescence Logic‐Signal‐Based Multiplex Detection of Nucleases with the Assembly of a Cationic Conjugated Polymer and Branched DNA

An energy‐transfer cascade is generated from a cationic conjugated polymer (PFP) and negatively charged, Y‐shaped DNA labeled with three dyes at its termini (fluorescein (Fl), Tex Red, and Cy5). Multistep fluorescence resonance energy transfer regulates the fluorescence intensities of PFP and the dyes. Different types of logic gates can be operated by observing the emission wavelengths of different dyes with multiplex nucleases as inputs.

     

Quencher-Delocalized Emission Strategy of AIEgen-Based Metal–Organic Framework for Profiling of Subcellular Glutathione

Distinguishing glutathione (GSH) level in different subcellular locations is critical for studying its antioxidant function in the signaling system. However, traditional methods for imaging subcellular GSH were achieved in isolated organelles or fixed cells. In this work, we report a quencher-delocalized emission strategy for in situ profiling of GSH at different subcellular locations in living cells. A nonemissive metal–organic framework (MOF) nanoprobe was designed with AIEgen as the linker and Cu^II as the node and quencher. The AIEgen in MOF structure was lightened up with green emission in a neutral environment due to partial Cu^II delocalization by competitive binding to GSH. Meanwhile, along with the protonation of AIEgen ligand under acidic environment, the AIEgen-based MOF could be completely dissociated in the presence of GSH to yield yellow emission. The two-channel ratiometric analysis of dual-colored emission of AIEgen-based MOF allows visualization of GSH in cytoplasm and lysosome in living cells, which is also able to report the drug effects on different subcellular GSH levels.     

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

外缘烷基链长对共轭有机小分子聚集行为及光电性质影响

改变分子化学结构和调控分子结构聚集态行为从而影响或改变材料的化学和物理性质, 是开发新型高效有机光电功能材料的重要手段. 在共轭有机分子外缘引入烷基链一般是为了改进材料溶解性能, 但近来的一些研究表明, 烷基链长对一些共轭有机小分子固态聚集行为和光电性质具有重要影响, 烷基链扮演着显著调控材料光电性质的“功能基团”作用. 本文以聚集诱导发光（aggregation-induced emission, AIE）/聚集强化荧光（aggregation enhanced emission, AEE）发射共轭有机小分子为重点, 对近年来有关烷基链长对共轭有机分子聚集形态和光电性质影响的一些典型事例进行评述, 旨在使人们在进行共轭有机分子设计合成及其结构与性能关系研究中能够关注烷基链的因素, 使烷基链变化作为功能导向晶态共轭有机材料设计合成及其可控制备的一种手段.     

A Freezing‐Induced Turn‐On Imaging Modality for Real‐Time Monitoring of Cancer Cells in Cryosurgery

Cryosurgery has attracted much attention for the treatment of tumors owing to its clear advantages. However, determining the volume of frozen tissues in real‐time remains a challenge, which greatly lowers the therapeutic efficacy of cryosurgery and hinders its broad application for the treatment of cancers. Herein, we report a freezing‐induced turn‐on strategy for the selective real‐time imaging of frozen cancer cells. As a type of aggregation‐induced emission (AIE) fluorogen, TABD‐Py molecules interact specifically with ice crystals and form aggregates at the ice/water interface. Consequently, bright fluorescent emission appears upon freezing. TABD‐Py molecules are enriched mostly in the cancer cells and exhibit high biocompatibility as well as low cytotoxicity; therefore, a freezing‐induced turn‐on imaging modality for cryosurgery is developed, which will certainly maximize the therapeutic efficacy of cryosurgery in treating tumors.     

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core–shell particles specifically recognizing glucuronic acid (GlcA) or N‐acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP‐GlcA and red QDs conjugated with MIP‐NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non‐imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.     

An AIE-Active Conjugated Polymer with High ROS-Generation Ability and Biocompatibility for Efficient Photodynamic Therapy of Bacterial Infections

New, biocompatible materials with favorable antibacterial activity are highly desirable. In this work, we develop a unique conjugated polymer featuring aggregation-induced emission (AIE) for reliable bacterial eradication. Thanks to the AIE and donor-π-acceptor structure, this polymer shows a high reactive oxygen species (ROS)-generation ability compared to a low-mass model compound and the common photosensitizer Chlorin E6. Moreover, the selective binding of pathogenic microorganisms over mammalian cells was found, demonstrating its biocompatibility. The effective growth inhibition of bacteria upon polymer treatment under light irradiation was validated in vitro and in vivo. Notably, the recovery from infection after treatment with our polymer is faster than that with cefalotin. Thus, this polymer holds great promise in fighting against bacteria-related infections in practical applications.     

Anisotropic optical properties in electroluminescent conjugated polymers based on grazing angle photoluminescence measurements

Grazing angle photoluminescence (GPL) originates from a waveguided light emitted at grazing angle to the substrate due to the total internal reflections, and the light emission is polarized with enhanced intensity at selective mode wavelength. GPL measurements reveal the optical anisotropy of luminescent conjugated polymers, in particular, the alignment of emitting dipoles from which emission occurs, in contrast to spectroscopic ellipsometry measurements that give the anisotropy in the absorption. Based on the GPL emission intensities and spectra, we investigate the anisotropic optical properties in electroluminescent poly(9,9'-di-n-octylfluorene-alt-benzothiadiazole) (F8BT) conjugated polymer thin films of different molecular weights (M(n) = 9-255 kg/mol), both in the pristine and annealed states. The optical anisotropy in F8BT films generally increases with molecular weight, suggesting that higher molecular weight polymers with longer chains are more likely to lie in-plane to the substrate. Upon annealing, high molecular weight F8BT films show even a higher degree of anisotropy, in contrast to low molecular weight F8BT films that become more isotropic. Annealing causes the polymer chains to rearrange and adopt a configuration in which the interchain exciton migration to better ordered low energy (LE) emissive states is strongly suppressed. We observe that the emissive states in F8BT are strongly affected by the local polymer chain arrangement, producing the less ordered high energy (HE) emissive states near the substrate interface where there is a higher degree of chain disorder and the LE states in the bulk of the film. When spin coated onto a quartz substrate precoated with a poly(styrenesulfonate)-doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) layer, films of F8BT show severe luminescence quenching near the PEDOT:PSS interface for both the LE and HE emissive states, but a selective quenching of the LE states in the bulk of the film. These observations have important implications for fabricating efficient electronic devices using conjugated polymers as an active material, since the performance of these devices will strongly depend on anisotropic optical properties of electroluminescent conjugated polymers.