A Deoxyuridine-Based Far-Red Emitting Viscosity Sensor.

A novel deoxyuridine (dU) benzothiazolium (BZ) derivative, referred to as dU-BZ, is reported that was synthesized via Sonogashira coupling reaction methodology. The deoxyuridine building block was introduced to enhance hydrophilicity, while an alkynylated benzothiazolium dye was incorporated for long wavelength absorption to reduce potential phototoxicity that is characteristic of using UV light to excite common fluorphores, better discriminate from native autofluorescence, and potentially facilitate deep tissue imaging. An impressive 30-fold enhancement of fluorescence intensity of dU-BZ was achieved upon increasing viscosity. Fluorescence quantum yields in 99% glycerol/1% methanol (v/v) solution as a function of temperature (293-343 K), together with viscosity-dependent fluorescence lifetimes and radiative and non-radiative rate constants in glycerol/methanol solutions (ranging from 4.8 to 950 cP) were determined. Both fluorescence quantum yields and lifetimes increased with increased viscosity, consistent with results predicted by theory. This suggests that the newly-designed compound, dU-BZ, is capable of functioning as a probe of local microviscosity, an aspect examined by in vitro bioimaging experiments.

RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vasculature imaging.

Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 µm in tumor tissue.

Deoxyribonucleoside-modified squaraines as near-IR viscosity sensors.

Deoxyribonucleoside-modified squaraines were synthesized by Sonogashira coupling reactions using an unsymmetrical, terminal alkynylated benzothiazolium squaraine dye. These non-natural nucleosides exhibited fluorescent 'turn-on' properties in viscous conditions with an enhancement of >300-fold. The viscosity-dependent fluorescence enhancement was attributed to a combination of hampering both molecular aggregation and intramolecular bond rotation of the squaraine probe. Fluorescence microscopy allowed visualization of highly viscous regions during various stages of cellular mitosis.

Selective cell death by photochemically induced pH imbalance in cancer cells.

Singlet oxygen sensitized photodynamic therapy (PDT) relies on the concentration of oxygen in the tissue to be treated. Most cancer lesions, however, have poor vasculature and, as a result, are hypoxic, significantly hindering PDT efficacies. An oxygen-independent PDT method may circumvent this limitation. To address this, we prepared sulfonium salts that produced a pH drop within HCT 116 cells via the generation of a photoacid within the cytosol. This process was driven by one- or two-photon absorption (1PA or 2PA) of the endocytosed photoacid generators (PAGs). One of these PAGs, which had a significantly lower dark cytotoxicity and was more efficient in generating a photoacid, effectively induced necrotic cell death in the HCT 116 cells. The data suggest that PAGs may be an attractive alternative PDT modality to selectively induce cell death in oxygen-deprived tissue such as tumors.

Long-wavelength, photostable, two-photon excitable BODIPY fluorophores readily modifiable for molecular probes.

Near-infrared (NIR) fluorescent probes are increasingly popular in biological imaging and sensing, as long-wavelength (650-900 nm) excitation and emission have the advantages of minimum photodamage, deep tissue penetration, and minimum interference from autofluorescence in living systems. Here, a series of long-wavelength BODIPY dyes SPC, DC-SPC, DPC, and DC-DPC are synthesized conveniently and efficiently. They exhibit excellent photophysical properties in far red to near-infrared region, including large extinction coefficients, high fluorescence quantum yields, good photostability, and reasonable two-photon absorption cross section. Comparison of single-molecular imaging confirms that DPC is a much more efficient and more photostable NIR fluorophore than the commonly used Cy5. Also importantly, two kinds of convenient functionalization sites have been reserved: the aryl iodide for organometallic couplings and the terminal alkyne groups for click reactions. Further derivatives DC-SPC-PPh3 exhibit specificity to localize in mitochondria. The introduction of triphenylphosphonium (TPP) moieties mediates its hydrophilic-lipophilic balance and makes DC-SPC-PPh3 appropriate for cell labeling. Their long-wavelength emission at ∼650 nm can efficiently avoid the spectral crosstalk with other probes emitting in the visible light region. Superior photostability, low cytotoxicity, and two-photon excitable properties demonstrate its utility as a standard colocalizing agent to estimate the other probes' local distribution.

Two-photon absorption and time-resolved stimulated emission depletion spectroscopy of a new fluorenyl derivative.

The synthesis, comprehensive linear photophysical characterization, two-photon absorption (2PA), steady-state and time-resolved stimulated emission depletion properties of a new fluorene derivative, (E)-1-(2-(di-p-tolylamino)-9,9-diethyl-9H-fluoren-7-yl)-3-(thiophen-2-yl)prop-2-en-1-one (1), are reported. The primary linear spectral properties, including excitation anisotropy, fluorescence lifetimes, and photostability, were investigated in a number of aprotic solvents at room temperature. The degenerate 2PA spectra of 1 were obtained with open-aperture Z-scan and two-photon induced fluorescence methods, using a 1 kHz femtosecond laser system, and maximum 2PA cross-sections of ∼400-600 GM were obtained. The nature of the electronic absorption processes in 1 was investigated by DFT-based quantum chemical methods implemented in the Gaussian 09 program. The one- and two-photon stimulated emission spectra of 1 were measured over a broad spectral range using a femtosecond pump-probe-based fluorescence quenching technique, while a new methodology for time-resolved fluorescence emission spectroscopy is proposed. An effective application of 1 in fluorescence bioimaging was demonstrated by means of one- and two-photon fluorescence microscopy images of HCT 116 cells containing dye encapsulated micelles.

[Effect of Matteuccia struthiopteris polysaccharides on systemic lupus erythematosus-like syndrome induced by Campylobacter jejuni in BALB/c mice].

Matteuccia struthiopteris is a nature plant, which contains a lot of potential active components. In the present study, we investigated the effect of polysaccharides extracted from Matteuccia struthiopteris on lupus-like syndrome induced by Campylobacter jejuni CJ-S131 in BALB/c mice. Mice were randomly divided into normal, model control, SLE model (vehicle treated), Matteuccia struthiopteris polysaccharides treated (30 and 15 mg x kg(-1)) groups and prednisone 5 mg x kg(-1) treated groups. The effect of Matteuccia struthiopteris polysaccharides (Ms) on weight and organ index of BALB/c mice was detected. Autoantibodies and total IgG production were measured by enzyme linked immunosorbent assay. Proteinuria was measured and kidneys were examined by light microscopy. Compared with SLE model group, treatment with Matteuccia struthiopteris polysaccharides 30 and 15 mg x kg(-1) reduced weight loss and Matteuccia struthiopteris polysaccharides 15 mg x kg(-1) reduced spleen swelling (P < 0.05). The increased production of autoantibodies and total immunoglobulin G (IgG) were also significantly inhibited. Matteuccia struthiopteris polysaccharides protected kidney against glomerular injury in BALB/c mice with reduced immunoglobulin deposition and lowered proteinuria (P < 0.01). Matteuccia struthiopteris polysaccharides had a protective effect on lupus-like syndrome induced by CJ-S131 in BALB/c mice.

Synthesis of Near-Infrared Fluorescent Two-Photon-Absorbing Fluorenyl Benzothiadiazole and Benzoselenadiazole Derivatives.

A series of dyes 2-5 based on 5-thienyl-2,1,3-benzothiadiazole and 5-thienyl-2,1,3-benzoselenadiazole cores were synthesized as near-infrared-emitting two-photon-absorbing fluorophores. Fluorescence maxima wavelengths as long as 714 nm and quantum yields as high as 0.67 were realized. The fluorescence quantum yields of dyes 2-4 were nearly constant, regardless of solvent polarity. These diazoles exhibited large Stokes shifts (>110 nm) and high two-photon figure of merit. Cells incubated on a 3D scaffold with probe 4 (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

Pegylated and nanoparticle-conjugated sulfonium salt photo triggers necrotic cell death.

Photodynamic therapy (PDT) processes involving the production of singlet oxygen face the issue of oxygen concentration dependency. Despite high oxygen delivery, a variety of properties related to metabolism and vascular morphology in cancer cells result in hypoxic environments, resulting in limited effectiveness of such therapies. An alternative oxygen-independent agent whose cell cytotoxicity can be remotely controlled by light may allow access to treatment of hypoxic tumors. Toward that end, we developed and tested both polyethylene glycol (PEG)-functionalized and hydrophilic silica nanoparticle (SiNP)-enriched photoacid generator (PAG) as a nontraditional PDT agent to effectively induce necrotic cell death in HCT-116 cells. Already known for applications in lithography and cationic polymerization, our developed oxygen-independent PDT, whether free or highly monodispersed on SiNPs, generates acid when a one-photon (1P) or two-photon (2P) excitation source is used, thus potentially permitting deep tissue treatment. Our study shows that when conjugated to SiNPs with protruding amine functionalities (SiNP-PAG9), such atypical PDT agents can be effectively delivered into HCT-116 cells and compartmentalize exclusively in lysosomes and endosomes. Loss of cell adhesion and cell swelling are detected when an excitation source is applied, suggesting that SiNP-PAG9, when excited via near-infrared 2P absorption (a subject of future investigation), can be used as a delivery system to selectively induce cell death in oxygen-deprived optically thick tissue.

Near-IR Two-Photon Fluorescent Sensor for K(+) Imaging in Live Cells.

A new two-photon excited fluorescent K(+) sensor is reported. The sensor comprises three moieties, a highly selective K(+) chelator as the K(+) recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (>52-fold) in detecting K(+) over other physiological metal cations. Upon binding K(+), the sensor switches from nonfluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K(+) sensing in living cells.