Unexpected omega-3 activities in intracellular lipolysis and macrophage foaming revealed by fluorescence lifetime imaging.

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo.

Short-Wave Infrared Quantum Dots with Compact Sizes as Molecular Probes for Fluorescence Microscopy.

Materials with short-wave infrared (SWIR) emission are promising contrast agents for in vivo animal imaging, providing high-contrast and high-resolution images of blood vessels in deep tissues. However, SWIR emitters have not been developed as molecular labels for microscopy applications in the life sciences, which require optimized probes that are bright, stable, and small. Here, we design and synthesize semiconductor quantum dots (QDs) with SWIR emission based on HgxCd1-xSe alloy cores red shifted to the SWIR by epitaxial deposition of thin HgxCd1-xS shells with a small band gap. By tuning alloy composition alone, the emission can be shifted across the visible-to-SWIR (VIR) spectra while maintaining a small and equal size, allowing direct comparisons of molecular labeling performance across a broad range of wavelength. After coating with click-functional multidentate polymers, the VIR-QD spectral series has high quantum yield in the SWIR (14-33%), compact size (13 nm hydrodynamic diameter), and long-term stability in aqueous media during continuous excitation. We show that these properties enable diverse applications of SWIR molecular probes for fluorescence microscopy using conjugates of antibodies, growth factors, and nucleic acids. A broadly useful outcome is a 10-55-fold enhancement of the signal-to-background ratio at both the single-molecule level and the ensemble level in the SWIR relative to visible wavelengths, primarily due to drastically reduced autofluorescence. We anticipate that VIR-QDs with SWIR emission will enable ultrasensitive molecular imaging of low-copy number analytes in biospecimens with high autofluorescence.

Multifunctional Conjugated Polymer Nanoparticles for Image-Guided Photodynamic and Photothermal Therapy.

A multifunctional theranostic platform based on conjugated polymer nanoparticles (CPNs) with tumor targeting, fluorescence detection, photodynamic therapy (PDT), and photothermal therapy (PTT) is developed for effective cancer imaging and therapy. Two conjugated polymers, poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)-ethyl)fluorenyldivinylene]-alt-4,7-(2,1,3-benzothiadiazole) with bright red emission and photosensitizing ability and poly[(4,4,9,9-tetrakis(4-(octyloxy)phenyl)-4,9-dihydro-s-indacenol-dithiophene-2,7-diyl)-alt-co-4,9-bis(thiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-thiadiazolo-quinoxaline] with strong near-infrared absorption and excellent photothermal conversion ability are co-loaded into one single CPN via encapsulation approach using lipid-polyethylene glycol as the matrix. The obtained co-loaded CPNs show sizes of around 30 nm with a high singlet oxygen quantum yield of 60.4% and an effective photothermal conversion efficiency of 47.6%. The CPN surface is further decorated with anti-HER2 affibody, which bestows the resultant anti-HER2-CPNs superior selectivity toward tumor cells with HER2 overexpression both in vitro and in vivo. Under light irradiation, the PDT and PTT show synergistic therapeutic efficacy, which provides new opportunities for the development of multifunctional biocompatible organic materials in cancer therapy.

Bovine Serum Albulmin Protein-Templated Silver Nanocluster (BSA-Ag13 ): An Effective Singlet Oxygen Generator for Photodynamic Cancer Therapy.

This paper reports a novel synthesis approach of bovine serum albumin (BSA) protein-templated ultrasmall (<2 nm) Ag nanocluster (NC) with strong singlet oxygen generation capacity for photodynamic therapy (PDT). An atomically precise BSA-Ag13 NC (i.e., 13 Ag atoms per cluster) is successfully synthesized for the first time by using NaOH-dissolved NaBH4 solution as the controlling reducing agent. The ubiquitous size of BSA-Ag13 NC results in unique behaviors of its photoexcited states as characterized by the ultrafast laser spectroscopy using time-correlated single photon counting and transient absorption techniques. In particular, triply excited states can be largely present in the excited BSA-Ag13 NC and readily sensitized molecular oxygen to produce singlet oxygen (1 O2 ) with a high quantum efficiency (≈1.26 using Rose Bengal as a standard). This value is much higher than its Au analogue (i.e., ≈0.07 for BSA-Au25 NC) and the commonly available photosensitizers. Due to the good cellular uptake and inherent biocompatibility imparted by the surface protein, BSA-Ag13 NC can be applied as an effective PDT agent in generating 1 O2 to kill cancer cell as demonstrated in this study.

Conjugated polymer microparticles for selective cancer cell image-guided photothermal therapy.

Nanotechnology has recently attracted great attention in biomedical research. Current nanoparticle approaches generally require further surface decoration with targeting ligands, peptides or proteins to achieve selective cancer imaging and therapy. This surface functionalization often complicates nanoparticles and leads to protein corona or varied nanoparticle uptake. In this work, we report a facile approach for selective cancer cell image-guided photothermal therapy by fabricating theranostic microparticles (MPs) using conjugated polymers (CPs) as the imaging and therapeutic agents. Through fine tuning of the backbone structures, we synthesized two CPs, poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9-di(thiophen-2-yl)-thiadiazoloquinoxaline] (PFTTQ) with high near infrared (NIR) molar absorptivity and poly(9,9-dihexylfluorene-alt-2,1,3-benzothiadiazole) (PFBT) with bright green emission. The two CPs were physically blended into single particles with ∼3 µm size, which was confirmed by scanning electron microscopy (SEM) and confocal fluorescence imaging. Although without any surface functionalization, the obtained CP MPs showed selective internalization into MCF-7 cancer cells over NIH-3T3 normal cells, while CP nanoparticles showed similar uptake into both cell lines. Moreover, the CP MPs could selectively kill MCF-7 cells upon NIR irradiation, which showed a half-maximal inhibitory concentration (IC50) of 30 µg mL-1 based on PFTTQ concentration.

A highly sensitive fluorescent light-up probe for real-time detection of the endogenous protein target and its antagonism in live cells.

Real-time detection and monitoring of cancer-related biomolecular interactions in live cells are of paramount importance for disease diagnostics and drug screening. Herein, we developed a target-specific fluorescent light-up probe for cellular detection of Mdm2, the key negative regulator of the p53 tumour suppressor protein. Conjugation of a uniquely designed fluorogen (TPECM) with aggregation induced-emission properties, to a specific p53-derived peptide (12.1Pep) targeting Mdm2, yielded a cell-permeable probe (TPECM-12.1Pep) with turn-on fluorescence properties for real-time live cell imaging of Mdm2. This specific light-up probe is almost non-fluorescent in its isolated state but is highly emissive upon binding to Mdm2, enabling quantitative detection of both Mdm2 and its antagonism. Using a model compound (Nutlin-3a), we demonstrate that the as-developed probes can be used to screen p53-Mdm2 inhibiting drug candidates, both in vitro and in cells. Furthermore, the probe activity can be accurately monitored in cells using a fluorescently activated cell sorting machine. These features will expedite research in the areas of drug discovery, clinical diagnostics and fundamental cell biology.

Biocompatible conjugated polymer nanoparticles for efficient photothermal tumor therapy.

Conjugated polymers (CPs) with strong near-infrared (NIR) absorption and high heat conversion efficiency have emerged as a new generation of photothermal therapy (PTT) agents for cancer therapy. An efficient strategy to design NIR absorbing CPs with good water dispersibility is essential to achieve excellent therapeutic effect. In this work, poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9-di(thiophen-2-yl)-thiadiazoloquinoxaline] (PFTTQ) is synthesized through the combination of donor-acceptor moieties by Suzuki polymerization. PFTTQ nanoparticles (NPs) are fabricated through a precipitation approach using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000 ) as the encapsulation matrix. Due to the large NIR absorption coefficient (3.6 L g(-1) cm(-1) ), the temperature of PFTTQ NP suspension (0.5 mg/mL) could be rapidly increased to more than 50 °C upon continuous 808 nm laser irradiation (0.75 W/cm(2) ) for 5 min. The PFTTQ NPs show good biocompatibility to both MDA-MB-231 cells and Hela cells at 400 µg/mL of NPs, while upon laser irradiation, effective cancer cell killing is observed at a NP concentration of 50 µg/mL. Moreover, PFTTQ NPs could efficiently ablate tumor in in vivo study using a Hela tumor mouse model. Considering the large amount of NIR absorbing CPs available, the general encapsulation strategy will enable the development of more efficient PTT agents for cancer or tumor therapy.

Fluorogen-peptide conjugates with tunable aggregation-induced emission characteristics for bioprobe design.

Fluorogens with aggregation-induced emission (AIE) characteristics are attracting intense research interest, and an AIE-peptide conjugate strategy has been reported for developing turn-on probes based on hydrophilic peptide ligands. To build a model also suitable for hydrophobic ligands, we propose to fine-tune the AIE characteristics for probe design. In this work, an iconic AIE fluorogen tetraphenylethene (TPE) was designed to conjugate with peptide fragments containing different numbers of aspartic acid (D) units. Relationships between the numbers of D and the hydrophilicity, optical properties, and aggregate sizes and the AIE characteristics of TPE-peptide conjugates were investigated carefully. Five carboxyl groups were found to be the threshold to "turn off" the fluorescence of TPE. As a proof-of-concept, TPE-SS-D5 containing a cleavable disulfide bond was synthesized for thiol turn-on detection. The validated tunable AIE characteristic offers new opportunities to design fluorescence turn-on probes based on hydrophobic recognition elements and AIE fluorogens.

Rational design of fluorescent light-up probes based on an AIE luminogen for targeted intracellular thiol imaging.

A water-soluble fluorescent light-up bioprobe based on a luminogen with aggregation-induced emission characteristics was developed for targeted intracellular thiol imaging.

Water-soluble bioprobes with aggregation-induced emission characteristics for light-up sensing of heparin.

Two water-soluble cationic fluorene-based fluorescent probes for heparin detection are designed and synthesized. A slight change in the molecular design results in two probes with opposite optical properties in their solution and aggregation states as well as a response to heparin in buffer solution. The probe with a propeller-like conformation exhibits aggregation-induced emission (AIE) characteristics and shows a green fluorescence enhancement upon interaction with heparin; in contrast, the probe with a more planar conformation has a fluorescence quenching response. A comprehensive study on heparin detection using the two probes was conducted, which revealed that the AIE probe shows a better performance than the aggregation-caused quenching (ACQ) probe in terms of sensitivity. The AIE probe integrated with graphene oxide (GO) further improves the heparin detection sensitivity and selectivity. The solution of AIE probe/GO emits strong green fluorescence only in the presence of heparin, which allows for light-up visual discrimination of heparin from its analogues such as chondroitin-4-sulfate and hyaluronic acid. Moreover, the linear light-up response of AIE probe/GO enables heparin quantification in the range of 0-13.2 µM with a detection limit of 10 nM, which is of practical importance for heparin monitoring during surgery or therapy.

A general approach to prepare conjugated polymer dot embedded silica nanoparticles with a SiO2@CP@SiO2 structure for targeted HER2-positive cellular imaging.

We report on a one-step synthesis of conjugated polymer (CP) embedded silica nanoparticles (NPs) with a SiO2@CP@SiO2 structure by combination of a precipitation method and a modified Stöber approach. Four types of CPs are employed to demonstrate the versatility of the developed strategy, yielding fluorescent silica NPs with emission across the visible spectrum. Field emission transmission electron microscopy investigation reveals that the entanglement between hydrophobic CPs and the aminopropyl groups of 3-aminopropyl triethoxysilane contributes to the successful encapsulation of CPs into a silica matrix. The synthesized NPs exhibit excellent physical stability and good photostability. In addition, they have amine groups on surfaces, which benefit further conjugation for biological applications. Through reaction with a peptide (GGHAHFG) that is specific to the HER2 receptor, the synthesized NPs have been successfully applied for targeted cellular imaging of HER2-overexpressed SKBR-3 breast cancer cells. Along with its high quantum yield and benign biocompatibility, the developed CP embedded silica NPs have great potential for applications in biological imaging.

Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing.

Long-term noninvasive cell tracing by fluorescent probes is of great importance to life science and biomedical engineering. For example, understanding genesis, development, invasion and metastasis of cancerous cells and monitoring tissue regeneration after stem cell transplantation require continual tracing of the biological processes by cytocompatible fluorescent probes over a long period of time. In this work, we successfully developed organic far-red/near-infrared dots with aggregation-induced emission (AIE dots) and demonstrated their utilities as long-term cell trackers. The high emission efficiency, large absorptivity, excellent biocompatibility, and strong photobleaching resistance of the AIE dots functionalized by cell penetrating peptides derived from transactivator of transcription proteins ensured outstanding long-term noninvasive in vitro and in vivo cell tracing. The organic AIE dots outperform their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for following biological processes such as carcinogenesis.

A bright far-red and near-infrared fluorescent conjugated polyelectrolyte with quantum yield reaching 25%.

We report a general strategy to design and synthesize a bright far-red and near-infrared fluorescent conjugated polyelectrolyte with a high quantum yield of 25% and a large Stokes shift of ~200 nm, which is ideal for cell imaging.

Lipid-PEG-folate encapsulated nanoparticles with aggregation induced emission characteristics: cellular uptake mechanism and two-photon fluorescence imaging.

Folate functionalized nanoparticles (NPs) that contain fluorogens with aggregation-induced emission (AIE) characteristics are fabricated to show bright far-red/near-infrared fluorescence, a large two-photon absorption cross section and low cytotoxicity, which are internalized into MCF-7 cancer cells mainly through caveolae-mediated endocytosis. One-photon excited in vivo fluorescence imaging illustrates that these AIE NPs can accumulate in a tumor and two-photon excited ex vivo tumor tissue imaging reveals that they can be easily detected in the tumor mass at a depth of 400 µm. These studies indicate that AIE NPs are promising alternatives to conventional TPA probes for biological imaging.

Glycosylated star-shaped conjugated oligomers for targeted two-photon fluorescence imaging.

A glucopyranose functionalized star-shaped oligomer, N-tris{4,4',4''-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-2-amido-2-deoxy-1-thio-ß-D-glucopyranose-hexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylamine (TVFVBN-S-NH(2)), is synthesized for two-photon fluorescence imaging. In water, TVFVBN-S-NH(2) self-assembles into nanoparticles with an average diameter of ∼49 nm and shows a fluorescence quantum yield of 0.21. Two-photon fluorescence measurements reveal that TVFVBN-S-NH(2) has a two-photon absorption cross-section of ∼1100 GM at 780 nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN-S-NH(2) with folic acid to yield TVFVBN-S-NH(2) FA with similar optical and physical properties as those for TVFVBN-S-NH(2). Cellular imaging studies reveal that TVFVBN-S-NH(2) FA has increased uptake by MCF-7 cells relative to that for TVFVBN-S-NH(2), due to specific interactions between folic acid and folate receptors on the MCF-7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water-soluble materials with a large two-photon absorption (TPA) cross-section for targeted cancer-cell imaging.

Specific detection of integrin αvß3 by light-up bioprobe with aggregation-induced emission characteristics.

Specific bioprobes with fluorescence turn-on response are highly desirable for high contrast biosensing and imaging. In this work, we developed a new generation bioprobe by integrating tetraphenylsilole, a fluorogenic unit with aggregation-induced emission (AIE) characteristic, with cyclic arginine-glycine-aspartic acid tripeptide (cRGD), a targeting ligand to integrin α(v)ß(3) receptor. Emission of the AIE probe is switched on upon its specific binding to integrin α(v)ß(3), which allows quantitative detection of integrin α(v)ß(3) in solution and real-time imaging of the binding process between cRGD and integrin α(v)ß(3) on cell membrane. The probe can be used for tracking integrin α(v)ß(3) and for identifying integrin α(v)ß(3)-positive cancer cells.

Conjugated polymer and gold nanoparticle co-loaded PLGA nanocomposites with eccentric internal nanostructure for dual-modal targeted cellular imaging.

Herein is reported the one-step synthesis of an integrated nanocomposite with eccentrically loaded 5 nm gold nanoparticles (Au NPs) and conjugated polymer of poly[9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorenyldivinylene-alt-4,7-(2,1,3,- benzothiadiazole) dibromide] (PFVBT). The nanocomposite is generated with surface-functionalized folic acid groups due to the matrix polymer of PLGA-PEG(2000) -folate used for encapsulation. The nanocomposite shows far-red fluorescence from PFVBT and scattering signal from Au NPs. Although Au NPs have been widely reported to quench the fluorescence of conjugated polymers, the PFVBT fluorescence is well maintained in the nanocomposite due to the eccentric location of Au NPs. The folic acid groups at the nanocomposite surface favor its cellular uptake by MCF-7 breast cancer cells, which have overexpressed folate receptors on the cell membranes. In conjugation with its low cytotoxicity, the folic-acid-functionalized nanocomposite has been successfully utilized for fluorescence and dark-field dual-modal targeted cellular imaging.