Conjugated Polymer Dots for Multiphoton Fluorescence Imaging

We report on the two-photon excited fluorescence of conjugated polymer dots (CPdots). As a new class of two-photon fluorescent probes, CPdots exhibit two-photon action cross sections as high as 2.0 × 105 GM, to our knowledge, the largest reported thus far for a nanoparticle. The cross section values are 3−4 orders of magnitude higher than those of conventional fluorescent dyes and an order of magnitude higher than those of inorganic quantum dots. Single particle fluorescence imaging was achieved using relatively low laser power

Tracking of Single Charge Carriers in a Conjugated Polymer Nanoparticle

The motion of individual charge carriers in organic nanostructures was tracked by fluorescence microscopy. A twinkling effect is observed in fluorescence microscopy of single conjugated polymer nanoparticles, that is, small displacements in the fluorescence spot of single nanoparticles of the conjugated polymer PFBT are observed over time. There is evidence that superquenching by the charge carrier induces a dark spot in the nanoparticle, which moves with the carrier, resulting in the observed displacements in the fluorescence. Zero-field mobilities of individual charge carriers consistent with highly trapped polarons were obtained from tracking experiments

Near-Infrared Fluorescent Dye-Doped Semiconducting Polymer Dots

Near-infrared (NIR) fluorescence sensing is desirable for in vivo biological measurements, but the method is currently limited by the availability of NIR fluorescent markers as well as by their poor performance, such as self-aggregation and dim fluorescence, in a physiological environment. To address this issue, this paper describes a NIR fluorescent polymer dot (Pdot) that emits at 777 nm. This Pdot was comparable in size to a water-soluble NIR quantum dot that emits at 800 nm (ITK Qdot800) but was about four times brighter and with a narrower emission peak. We formed the NIR Pdot by doping the NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), into the matrix of poly (9,9-dioctylfluorene-co-benzothiadiazole) (PFBT) as the Pdot formed using a nanoscale precipitation technique. Free molecules of NIR775 aggregate in aqueous solution, but encapsulating them into the hydrophobic Pdot matrix effectively introduced them into aqueous solution for use in biological studies. Most importantly, the brightness of NIR775 was dramatically enhanced because of the excellent light-harvesting ability of PFBT and the very efficient energy transfer from PFBT to NIR775. We anticipate this bright NIR Pdot will be useful in biological measurements and cellular imaging where strong NIR emission is beneficial

<i>In Vivo</i> Imaging of Exosomes Labeled with NIR-II Polymer Dots in Liver-Injured Mice

Mesenchymal stem cell-derived exosomes (MSC-Exos) are emerging as a promising platform for treating various intractable diseases and organ injuries. Monitoring their migration, homing, and therapeutic capability in vivo is essential to develop exosome-based theranostics. Here, we designed fluorescent semiconductor polymer dots (Pdots) in the second near-infrared window (NIR-II) for bright labeling and tracking of MSC-Exos. Glucose-coated Pdots (Pdots-Glu) were able to label MSC-Exos without changing their biological properties. The NIR-II fluorescent Pdots allow for high labeling brightness and long-term in vivo tracking of MSC-Exos. We investigated the biodistributions and therapeutic functions of these labeled MSC-Exos in liver-resected mice. In vivo and ex vivo imaging demonstrated that the Pdot-labeled MSC-Exos injected via the tail vein mainly accumulated in the residual liver tissue. In terms of the therapeutic effect, MSC-Exos may accelerate postoperative liver function recovery by inhibiting inflammatory responses, promoting cell proliferation, and resisting apoptosis. Our results indicated that MSC-Exos therapeutic systems hold promising applications in liver regenerative medicine

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

Ratiometric Temperature Sensing with Semiconducting Polymer Dots

This communication describes ultrabright single-nanoparticle ratiometric temperature sensors based on semiconducting polymer dots (Pdots). We attached the temperature sensitive dyeRhodamine B (RhB), whose emission intensity decreases with increasing temperaturewithin the matrix of Pdots. The as-prepared Pdot-RhB nanoparticle showed excellent temperature sensitivity and high brightness because it took advantage of the light harvesting and amplified energy transfer capability of Pdots. More importantly, the Pdot-RhB nanoparticle showed ratiometric temperature sensing under a single wavelength excitation and has a linear temperature sensing range that matches well with the physiologically relevant temperatures. We employed Pdot-RhB for measuring intracellular temperatures in a live-cell imaging mode. The exceptional brightness of Pdot-RhB allows this nanoscale temperature sensor to be used also as a fluorescent probe for cellular imaging

Development of Ultrabright Semiconducting Polymer Dots for Ratiometric pH Sensing

Semiconducting polymer-based nanoparticles (Pdots) have recently emerged as a new class of ultrabright probes for biological detection and imaging. This paper describes the development of poly(2,5-di(3′,7′-dimethyloctyl)phenylene-1,4-ethynylene) (PPE) Pdots as a platform for designing Förster resonance energy transfer (FRET)-based ratiometric pH nanoprobes. We describe and compare three routes for coupling the pH-sensitive dye, fluorescein, to PPE Pdots, which is a pH-insensitive semiconducting polymer. This approach offers a rapid and robust sensor for pH determination using the ratiometric methodology where excitation at a single wavelength results in two emission peaks, one that is pH sensitive and the other one that is pH insensitive for use as an internal reference. The linear range for pH sensing of the fluorescein-coupled Pdots is between pH 5.0 and 8.0, which is suitable for most cellular studies. The pH-sensitive Pdots show excellent reversibility and stability in pH measurements. In this paper, we use them to measure the intracellular pH in HeLa cells following their uptake by endocytosis, thus demonstrating their utility for use in cellular and imaging experiments

Nanoscale 3D Tracking with Conjugated Polymer Nanoparticles

Small (∼15 nm diameter), highly fluorescent conjugated polymer nanoparticles were evaluated for nanoscale 2D and 3D tracking applications. Nanoparticles composed of conjugated polymers possess high absorption cross sections, high radiative rates, and low or moderate aggregation quenching, resulting in extraordinarily high fluorescent brightness. The bright fluorescence (∼200 000 photons detected per particle per 20 ms exposure) yields a theoretical particle tracking uncertainty of less than 1 nm. A lateral tracking uncertainty of 1−2 nm was determined from analysis of trajectories of fixed and freely diffusing particles. Axial (Z) position information for 3D particle tracking was obtained by defocused imaging. Nanoscale tracking of single particles in fixed cells was demonstrated, and a range of complex behaviors, possibly due to binding/unbinding dynamics, were observed

Incorporation of Porphyrin to π‑Conjugated Backbone for Polymer-Dot-Sensitized Photodynamic Therapy

The photosensitizers used in photodynamic therapy are mainly based on porphyrin derivatives. However, clinical applications encounter several limitations regarding photosensitizers such as their low absorption coefficients, poor water-solubility, and leaching from delivery carriers. Here, we describe covalent incorporation of porphyrin in conjugated polymer backbone for development of efficient polymer-dot photosensitizer. Spectroscopic characterizations revealed that the light-harvesting polymer dominantly transfer the excitation energy to the porphyrin unit, yielding efficient singlet oxygen generation for photodynamic therapy. The polymer dots (Pdots) also possess excellent stability that overcomes the photosensitizer leaching problem as encountered in other nanoparticle carriers. In vitro cytotoxicity and photodynamic efficacy of the Pdots were evaluated in MCF-7 cells by in vitro assay, indicating that the Pdots can efficiently damage cancer cells. In vivo photodynamic therapy by using the Pdots was further investigated with xenograft tumors in Balb/c nude mice, which show that the tumors were significantly inhibited or eradicated in certain cases. The high-yield singlet oxygen generation and excellent stability of porphyrin-incorporated Pdots are promising for photodynamic treatment of malignant tumors

Bioconjugation of IgG Secondary Antibodies to Polymer Dots for Multicolor Subcellular Imaging

Multiplexed subcellular imaging has attracted widespread attention in biology and medicine. Because the fluorescence brightness and stability of organic dyes and fluorescent proteins are not sufficient, the development of fluorescent nanoparticles is of broad interest. Semiconducting polymer dots (Pdots) exhibit prominent luminescence properties. However, the Pdot-IgG bioconjugates are largely unexplored in biological imaging. Here, we developed multicolor Pdots covalently conjugated with IgG secondary antibodies for specific labeling of different subcellular structures. By comparing the two functional polymers, we found that the functionalization by poly­(styrene-co-maleic anhydride) (PSMA) could reliably produce Pdot-IgG conjugates for specific subcellular labeling, while those by a comb-like amphiphilic polymer show strong nonspecific binding in an intracellular environment. In addition, we minimize nonspecific adsorption of the Pdot bioconjugates by using bovine serum albumin and poly­(ethylene glycol) as passivation agents, respectively. We finally demonstrate that the Pdot-PSMA-IgGs hold great potential in multiplexed visualization of the subcellular structure