Enhancement of gemcitabine sensitivity in intrahepatic cholangiocarcinoma through Saikosaponin-a mediated modulation of the p-AKT/BCL-6/ABCA1 axis.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) remains a significant challenge in cancer therapy, especially due to its resistance to established treatments like Gemcitabine, necessitating novel therapeutic approaches. METHODS: This study utilized Gemcitabine-resistant cell lines, patient-derived organotypic tumor spheroids (PDOTs), and patient-derived xenografts (PDX) to evaluate the effects of Saikosaponin-a (SSA) on ICC cellular proliferation, migration, apoptosis, and its potential synergistic interaction with Gemcitabine. Techniques such as transcriptome sequencing, Luciferase reporter assays, and molecular docking were employed to unravel the molecular mechanisms. RESULTS: SSA exhibited antitumor effects in both in vitro and PDX models, indicating its considerable potential for ICC treatment. SSA markedly inhibited ICC progression by reducing cellular proliferation, enhancing apoptosis, and decreasing migration and invasion. Crucially, it augmented Gemcitabine's efficacy by targeting the p-AKT/BCL6/ABCA1 signaling pathway. This modulation led to the downregulation of p-AKT and suppression of BCL6 transcriptional activity, ultimately reducing ABCA1 expression and enhancing chemosensitivity to Gemcitabine. Additionally, ABCA1 was validated as a predictive biomarker for drug resistance, with a direct correlation between ABCA1 expression levels and the IC50 values of various small molecule drugs in ICC gene profiles. CONCLUSION: This study highlights the synergistic potential of SSA combined with Gemcitabine in enhancing therapeutic efficacy against ICC and identifies ABCA1 as a key biomarker for drug responsiveness. Furthermore, the introduction of the novel PDOTs microfluidic model provides enhanced insights into ICC research. This combination strategy may provide a novel approach to overcoming treatment challenges in ICC.

Dual-signal ratiometric electrochemiluminescence biosensor based on Au NPs-induced low-potential emission of PFO Pdots and LSPR-ECL mechanism for ultra-sensitive detection of microRNA-141.

In this study, we have for the first time constructed a ratiometric ECL biosensor for the ultrasensitive detection of microRNAs (miRNAs) using gold nanoparticles (Au NPs) to trigger both the low-potential emission from conjugated polymer poly(9,9-dioctylfluorene-2,7-diyl) dots (PFO Pdots) and the LSPR-ECL effect with sulfur-doped boron nitride quantum dots (S-BN QDs). PFO Pdots were first applied to the Au NPs-modified electrode, followed by covalent binding to capture the hairpin H1. Immediately thereafter, a small amount of miRNA-141 was able to generate a large amount of output DNA (OP) by traversing the target cycle. OP, H3-S-BN QDs, and H4-glucose oxidase (H4-GOD) were then added sequentially to the Au NPs-modified electrode surface, and the hybridization chain reaction (HCR) was initiated. This resulted in the introduction of a large amount of GOD into the system, which catalyzed the in situ formation of the co-reactant hydrogen peroxide (H2O2) from the substrate glucose. Due to the electron transfer effect, the production of H2O2 led to the ECL quenching of PFO Pdots. Meanwhile, H2O2 served as a co-reactant of S-BN QDs, resulting in strong ECL emission of S-BN QDs at the cathode. Furthermore, the cathodic ECL intensity of S-BN QDs was further enhanced by an LSPR-ECL mechanism between Au NPs and S-BN QDs. By measuring the ratio of ECL intensities at two excitation potentials, this approach could provide sensitive and reliable detection of miRNA-141 in the range of 0.1 fM ∼10 nM, with a detection limit of 0.1 fM.

Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

Resonant Raman-Active Polymer Dot Barcodes for Multiplex Cell Mapping.

Resonance Raman spectroscopy is an efficient tool for multiplex imaging because of the narrow bandwidth of the electronically enhanced vibrational signals. However, Raman signals are often overwhelmed by concurrent fluorescence. In this study, we synthesized a series of truxene-based conjugated Raman probes to show structure-specific Raman fingerprint patterns with a common 532 nm light source. The subsequent polymer dot (Pdot) formation of the Raman probes efficiently suppressed fluorescence via aggregation-induced quenching and improved the dispersion stability of particles without leakage of Raman probes or particle agglomeration for more than 1 year. Additionally, the Raman signal amplified by electronic resonance and increased probe concentration exhibited over 103 times higher relative Raman intensities versus 5-ethynyl-2'-deoxyuridine, enabling successful Raman imaging. Finally, multiplex Raman mapping was demonstrated with a single 532 nm laser using six Raman-active and biocompatible Pdots as barcodes for live cells. Resonant Raman-active Pdots may suggest a simple, robust, and efficient way for multiplex Raman imaging using a standard Raman spectrometer, suggesting the broad applicability of our strategy.

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.

Construction of Effective Nanosensor by Combining Semiconducting Polymer Dots with Diphenylcarbazide for Specific Recognition of Trace Cr (VI) Ion in Water and Vitro.

Hexavalent chromium (Cr (VI)) ion, as highly toxic environmental pollution, severely endangers the ecological environment and public health. Herein, a fluorescent nanosensor (PFO-DPC) was constructed by combining semiconducting polymer dots with diphenylcarbazide (DPC) for sensing Cr (VI) ion in aqueous solution and living cells. DPC and poly (styrene-co-maleic anhydride) (PSMA) polymer mixed with polyfluorene (PFO) were utilized for selectively indicating Cr (VI) ion and improving the efficiency of detection, respectively. The presence of Cr (VI) ion effectively turned off the blue and green fluorescence of PFO-DPC in the aqueous environment, and the fluorescence quenching efficiency exhibited a good linear relationship between the range of 0.0 to 2.31 nM (R2 = 0.983) with a limit of detection (LOD) of 0.16 nM. The mechanism of fluorescence quenching could possibly be attributed to the internal filtration effect (IFE). Additionally, PFO-DPC showed a satisfactory performance in monitoring intracellular Cr (VI) ion. Our results indicate that the sensor is promising in various applications.

Ultrabright Pdots with a Large Absorbance Cross Section and High Quantum Yield.

Semiconducting polymer dots (Pdots) are increasingly used in biomedical applications due to their extreme single-particle brightness, which results from their large absorption cross section (σ). However, the quantum yield (Φ) of Pdots is typically below 40% due to aggregation-induced self-quenching. One approach to reducing self-quenching is to use FRET between the donor (D) and acceptor (A) groups within a Pdot; however, Φ values of FRET-based Pdots remain low. Here, we demonstrate an approach to achieve ultrabright FRET-based Pdots with simultaneously high σ and Φ. The importance of self-quenching was revealed in a non-FRET Pdot: adding 30 mol % of a nonabsorbing polyphenyl to a poly(9,9-dioctylfluorene) (PFO) Pdot increased Φ from 13.4 to 71.2%, yielding an ultrabright blue-emitting Pdot. We optimized the brightness of FRET-based Pdots by exploring different D/A combinations and ratios with PFO and poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-phenylene)] (PFP) as donor polymers and poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-(1,4-phenylene)] (PFPV) and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT) as acceptor polymers, with a fixed concentration of poly(styrene-co-maleic anhydride) as surfactant polymer. Ultrabright blue-emitting Pdots possessing high Φ (73.1%) and σ (σR = σabs/σall, 97.5%) were achieved using PFP/PFPV Pdots at a low acceptor content (A/[D + A], 2.5 mol %). PFP/PFPV Pdots were 1.8 times as bright as PFO/PFPV Pdots due to greater coverage of acceptor absorbance by donor emission─a factor often overlooked in D/A pair selection. Ultrabright green-emitting PFO Pdots (Φ = 76.0%, σR = 92.5%) were obtained by selecting an acceptor (PFBT) with greater spectral overlap with PFO. Ultrabright red-emitting Pdots (Φ = 64.2%, σR = 91.0%) were achieved by blending PFO, PFBT, and PFTBT to create a cascade FRET Pdot at a D:A1:A2 molar ratio of 61:5:1. These blue, green, and red Pdots are among the brightest Pdots reported. This approach of using a small, optimized amount of FRET acceptor polymer with a large donor-acceptor spectral overlap can be generalized to produce ultrabright Pdots with emissions that span the visible spectrum.

Gadolinium and Polythiophene Functionalized Polyurea Polymer Dots as Fluoro-Magnetic Nanoprobes.

A rapid and one-pot synthesis of poly 3-thiopheneacetic acid (PTAA) functionalized polyurea polymer dots (Pdots) using polyethyleneimine and isophorone diisocyanate is reported. The one-pot mini-emulsion polymerization technique yielded Pdots with an average diameter of ~20 nm. The size, shape, and concentration of the surface functional groups could be controlled by altering the synthesis parameters such as ultrasonication time, concentration of the surfactant, and crosslinking agent, and the types of isocyanates utilized for the synthesis. Colloidal properties of Pdots were characterized using dynamic light scattering and zeta potential measurements. The spherical geometry of Pdots was confirmed by scanning electron microscopy. The Pdots were post-functionalized by 1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid for chelating gadolinium nanoparticles (Gd3+) that provide magnetic properties to the Pdots. Thus, the synthesized Pdots possess fluorescent and magnetic properties, imparted by PTAA and Gd3+, respectively. Fluorescence spectroscopy and microscopy revealed that the synthesized dual-functional Gd3+-Pdots exhibited detectable fluorescent signals even at lower concentrations. Magnetic levitation experiments indicated that the Gd3+-Pdots could be easily manipulated via an external magnetic field. These findings illustrate that the dua- functional Gd3+-Pdots could be potentially utilized as fluorescent reporters that can be magnetically manipulated for bioimaging applications.

Hydrophobic and Hydrophilic Conjugated Polymer Dots as Binary Photocatalysts for Enhanced Visible-Light-Driven Hydrogen Evolution through Förster Resonance Energy Transfer.

Organic semiconducting polymers exhibited promising photocatalytic behavior for hydrogen (H2) evolution, especially when prepared in the form of polymer dots (Pdots). However, the Pdot structures were formed using common nonconjugated amphiphilic polymers, which have a negative effect on charge transfer between photocatalysts and reactants and are unable to participate in the photocatalytic reaction. This study presents a new strategy for constructing binary Pdot photocatalysts by replacing the nonconjugated amphiphilic polymer typically employed in the preparation of polymer nanoparticles (Pdots) with a low-molecular-weight conjugated polyelectrolyte. The as-prepared polyelectrolyte/hydrophobic polymer-based binary Pdots truly enhance the electron transfer between the Pt cocatalyst and the polymer photocatalyst with good water dispersibility. Moreover, unlike the nonconjugated amphiphilic polymer, the photophysics and mechanism of this photocatalytic system through time-correlated single-photon counting (TCSPC) and transient absorption (TA) measurements confirmed the Förster resonance energy transfer (FRET) between the polyelectrolyte as a donor and the hydrophobic polymer as an acceptor. As a result, the designated binary Pdot photocatalysts significantly enhanced the hydrogen evolution rate (HER) of 43 900 µmol g-1 h-1 (63.5 µmol h-1, at 420 nm) for PTTPA/PFTBTA Pdots under visible-light irradiation.

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.

Recent Developments in Semiconducting Polymer Dots for Analytical Detection and NIR-II Fluorescence Imaging.

In recent years, semiconducting polymer dots (Pdots) have attracted enormous attention in applications from fundamental analytical detection to advanced deep-tissue bioimaging due to their ultrahigh fluorescence brightness with excellent photostability and minimal cytotoxicity. Pdots have therefore been widely adopted for a variety types of molecular sensing for analytical detection. More importantly, the recent development of Pdots for use in the optical window between 1000 and 1700 nm, popularly known as the "second near-infrared window" (NIR-II), has emerged as a class of optical transparent imaging technology in the living body. The advantages of the NIR-II region over the traditional NIR-I (700-900 nm) window in fluorescence imaging originate from the reduced autofluorescence, minimal absorption and scattering of light, and improved penetration depths to yield high spatiotemporal images for biological tissues. Herein, we discuss and summarize the recent developments of Pdots employed for analytical detection and NIR-II fluorescence imaging. Starting with their preparation, the recent developments for targeting various analytes are then highlighted. After that, the importance of and latest progress in NIR-II fluorescence imaging using Pdots are reported. Finally, perspectives and challenges associated with the emergence of Pdots in different fields are given.

A novel electrochemiluminescence biosensor based on the self-ECL emission of conjugated polymer dots for lead ion detection.

The poly[(9,9-dioctylfuorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT) was carboxyl-functionalized to prepare polymer dots (C-PFBT Pdots), which served as a self-ECL emitter for producing an extraordinary ECL signal without any exogenous coreactants. The C-PFBT Pdots-modified electrode captured the substrate DNA and further hybridized with a ferrocene (Fc)-labeled DNA. The ECL emission of C-PFBT Pdots was quenched by Fc (a signal off state). After the DNAzyme was added, the DNAzyme-substrate hybrids were formed through hybridizing between DNAzyme and substrate and the Fc-labeled DNA was released. In the presence of target Pb2+, the DNAzyme-substrate hybrids could be specifically recognized and cleaved to release the DNAzyme and Pb2+. Ultimately, the released DNAzyme would further hybridize with the substrate for producing the DNAzyme-substrate hybrids and then were cleaved by the released Pb2+. As a result, the DNA walking machine was generated and the substantial Fc was away from C-PFBT Pdots to obtain a signal on state. Such a strategy achieved a sensitive detection of Pb2+ and the detection limit was as low as 0.17 pM. Moreover, making this ECL biosensor for an intracellular Pb2+ detecting, a convincing performance was achieved. The self-ECL emitter C-PFBT Pdots combining with the quencher Fc provided a new strategy and platform for constructing a coreactant-free ECL assay.

An ultrasensitive sensing platform for microRNA-155 based on H2O2 quenched hydroxide-dependent ECL emission of PFO Pdots.

A strong hydroxide (OH-)-dependent ECL emission of carboxyl functionalized-poly (9,9-di-n-octylfluorenyl-2,7-diyl) polymer dots (PFO Pdots) was observed at +1.25 V, which is significantly stronger than the emission at +1.95 V reported in previous work. Moreover, hydrogen peroxide (H2O2) can efficiently quench OH--dependent ECL emission of PFO Pdots. Based on this discovery, a signal "off-on" ECL biosensing platform for microRNA-155 (miRNA-155) was developed. Firstly, PFO Pdots were modified onto the electrode to capture DNA duplex track-locker. In the presence of H2O2 in the test solution, the ECL signal of PFO Pdots was quenched to obtain a signal-off state. Subsequently, the DNA walker produced through the target miRNA-155-triggered catalytic hairpin assembly (CHA) walked along the DNA duplex track-locker to output amounts of G-rich short chain, forming a hemin/G-quadruplex. With the consumption of H2O2 by hemin/G-quadruplex, the ECL signal would be restored to a signal-on state, thus achieving an ultrasensitive detection of miRNA-155. The detection limit was low as 12.2 aM. Furthermore, our proposed biosensor demonstrated a tremendous selectivity and admirable stability, and exhibited a satisfactory performance for determinating intracellular miRNA-155. The integration of excellent ECL performance of PFO Pdots without any exogenous species or dissolved O2 as co-reactant and a highly efficient quenching effect of H2O2 on such an ECL emission will provide an attractive ECL platform for bioanalysis and clinical diagnosis.

Cyanine-Based Polymer Dots with Long-Wavelength Excitation and Near-Infrared Fluorescence beyond 900 nm for In Vivo Biological Imaging.

Bioimaging in the near-infrared window is of great importance to study the dynamic processes in vivo with deep penetration, high spatiotemporal resolution, and minimal tissue absorption, scattering, and autofluorescence. In spite of the huge progress on the synthesis of small organic fluorophores and inorganic nanomaterials with emissions beyond 900 nm, it remains a tough challenge to synthesize semiconducting polymers with fluorescence over this region. Here, we synthesized a series of heptamethine cyanine-based polymers with both absorption and emission in the near-infrared region. We prepared these polymers as semiconducting polymer dots (Pdots) in pure water with great biocompatibility. The fluorescence quantum yield of the Pdots can be as high as 14% with a full width at half-maximum of 53 nm, and their single-particle brightness is more than 20 times higher than commercial quantum dots or ∼300 times brighter than Food and Drug Administration (FDA)-approved indocyanine green (ICG) dyes. We further demonstrated the use of cyanine-based Pdots for specific cellular labeling and long-term tumor targeting in mice. We anticipate that these cyanine-based ultrabright Pdots could open up an avenue for next generations of near-infrared fluorescent agents.

Ratiometric pH Sensing and Imaging in Living Cells with Dual-Emission Semiconductor Polymer Dots.

Polymer dots (Pdots) represent newly developed semiconductor polymer nanoparticles and exhibit excellent characteristics as fluorescent probes. To improve the sensitivity and biocompatibility of Pdots ratiometric pH biosensors, we synthesized 3 types of water-soluble Pdots: Pdots-PF, Pdots-PP, and Pdots-PPF by different combinations of fluorescent dyes poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), poly[(9,9-dioctyl-fluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadazole)] (PFBT), and fluorescein isothiocyanate (FITC). We found that Pdots-PPF exhibits optimal performance on pH sensing. PFO and FITC in Pdots-PPF produce pH-insensitive (λ = 439 nm) and pH-sensitive (λ = 517 nm) fluorescence respectively upon a single excitation at 380 nm wavelength, which enables Pdots-PPF ratiometric pH sensing ability. Förster resonance energy transfer (FRET) together with the use of PFBT amplify the FITC signal, which enables Pdots-PPF robust sensitivity to pH. The emission intensity ratio (I517/I439) of Pdots-PPF changes linearly as a function of pH within the range of pH 3.0 to 8.0. Pdots-PPF also possesses desirable reversibility and stability in pH measurement. More importantly, Pdots-PPF was successfully used for cell imaging in Hela cells, exhibiting effective cellular uptake and low cytotoxicity. Our study suggests the promising potential of Pdots-PPF as an in vivo biomarker.

Dual-readout immunosensor constructed based on brilliant photoelectrochemical and photothermal effect of polymer dots for sensitive detection of sialic acid.

A delicate dual-readout immunosensor based on tetraphenylporphyrin-polymer dots (TPP-Pdots) with brilliant photoelectrochemical and photothermal performance was first successfully fabricated for the ultrasensitive detection of sialic acid (SA). Herein, TPP-Pdots with good biocompatibility, extraordinary light-harvesting ability and excellent photothermal conversion efficiency was used to capture SA antibody as dual-functional bioprobe for generating photocurrent and temperature signal. Furthermore, the large surface and morphology-mediated of rutile-TiO2 (R-TiO2) was beneficial to load amounts of TPP-Pdots for improved PEC signal and photothermal signal. Importantly, the temperature readout resulted from the variation of target concentration could be easily obtained by a universal thermometer which was time-saving and cost-saving. Under the optimized experimental conditions, the photocurrent densities and temperature changes proportionally increased with the increasing of SA concentrations from 3.5 × 10-5 ng/mL to 35 ng/mL (R = 0.996). Impressively, the dual-readout approach proposed here not only featured with good accuracy and high sensitivity for SA detection, but also paved the way for the development of a dual-readout immunoassay based on PEC biosensor.

'Imperfect' conjugated polymer nanoparticles from MEH-PPV for bioimaging and Fe(III) sensing.

A simple and effective method was reported for the preparation from MEH-PPV of conjugated polymer nanoparticles (Pdots) that are water-soluble and well dispersed. The as-prepared Pdots show bright orange fluorescence at a quantum yield up to 32.37%. The fluorescence intensity of Pdots can be quenched with good selectively by the successive addition of Fe(3+) . In addition, the as-obtained Pdots were applied to the imaging of HeLa cells, and exhibited low cytotoxicity and excellent biocompatibility.