High-resolution light microscopy using luminescent nanoparticles.

This review presents recent progress in the development of the luminescent nanoparticles for confocal and multiphoton microscopy. Four classes of nanomaterials are discussed: (1) silica-based nanoparticles doped with fluorescent molecules, (2) gold nanoparticles, (3) semiconductor nanocrystals (quantum dots/rods), and (4) nanophosphors. Special considerations are given to recently developed imaging nanoprobes, such as (1) organically modified silica (ORMOSIL) nanoparticles doped with two-photon absorbing fluorophores, which exhibit aggregation-enhanced fluorescence (AEF), and (2) nanophosphors (ceramic nanoparticles containing luminescent lanthanoid ions). Advantages and disadvantages of every class of nanomaterials and their specific applications are briefly discussed.

Hyperbranched polysiloxysilane nanoparticles: surface charge control of nonviral gene delivery vectors and nanoprobes.

New hyperbranched polysiloxysilane (HBPS) materials containing terminal carboxylic acid and quaternary ammonium groups were designed and synthesized to obtain fluorescent-dye-encapsulated nanoparticles. These polymers exhibited desirable characteristics, including amphiphilicity for nanoparticle formation, and contained various terminal groups for surface-charge control on the nanoparticles or for further bioconjugation for targeted imaging. Nanoprobes composed of polysiloxysilane nanoparticles encapsulating two-photon dyes were also prepared for optical bioimaging with controlled surface charge density (zeta potential) for modulation of cellular uptake. Intracellular delivery of these structurally similar polysiloxysilane nanoparticles, with substantially different surface charges, was investigated using confocal and two-photon fluorescence microscopy as well as flow cytometry. Finally, the use of these nanoparticles as efficient gene delivery vectors was demonstrated by means of in vitro transfection study using beta-galactosidase plasmid and pEGFP-N1 plasmid and the most efficient combination was obtained using HBPS-CN30:70.

New method for delivering a hydrophobic drug for photodynamic therapy using pure nanocrystal form of the drug.

A carrier-free method for delivery of a hydrophobic drug in its pure form, using nanocrystals (nanosized crystals), is proposed. To demonstrate this technique, nanocrystals of a hydrophobic photosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH), have been synthesized using the reprecipitation method. The resulting drug nanocrystals were monodispersed and stable in aqueous dispersion, without the necessity of an additional stabilizer (surfactant). As shown by confocal microscopy, these pure drug nanocrystals were taken up by the cancer cells with high avidity. Though the fluorescence and photodynamic activity of the drug were substantially quenched in the form of nanocrystals in aqueous suspension, both these characteristics were recovered under in vitro and in vivo conditions. This recovery of drug activity and fluorescence is possibly due to the interaction of nanocrystals with serum albumin, resulting in conversion of the drug nanocrystals into the molecular form. This was confirmed by demonstrating similar recovery in presence of fetal bovine serum (FBS) or bovine serum albumin (BSA). Under similar treatment conditions, the HPPH in nanocrystal form or in 1% Tween-80/water formulation showed comparable in vitro and in vivo efficacy.

Organically modified silica nanoparticles co-encapsulating photosensitizing drug and aggregation-enhanced two-photon absorbing fluorescent dye aggregates for two-photon photodynamic therapy.

We report energy-transferring organically modified silica nanoparticles for two-photon photodynamic therapy. These nanoparticles co-encapsulate two-photon fluorescent dye nanoaggregates as an energy up-converting donor and a photosensitizing PDT drug as an acceptor. They combine two features: (i) aggregation-enhanced two-photon absorption and emission properties of a novel two-photon dye and (ii) nanoscopic fluorescence resonance energy transfer between this nanoaggregate and a photosensitizer, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide. Stable aqueous dispersions of the co-encapsulating nanoparticles (diameter < or = 30 nm) have been prepared in the nonpolar interior of micelles by coprecipitating an organically modified silica sol with the photosensitizer and an excess amount of the two-photon dye which forms fluorescent aggregates by phase separation from the particle matrix. Using a multidisciplinary nanophotonic approach, we show: (i) indirect excitation of the photosensitizer through efficient two-photon excited intraparticle energy transfer from the dye aggregates in the intracellular environment of tumor cells and (ii) generation of singlet oxygen and in vitro cytotoxic effect in tumor cells by photosensitization under two-photon irradiation.

Dye-concentrated organically modified silica nanoparticles as a ratiometric fluorescent pH probe by one- and two-photon excitation.

Basic dye-concentrated nanoparticles (approximately 33 nm in diameter) show fluorescence-based ratiometric pH response, by one- and two-photon excitations, with improved proton sensing ability (pKa approximately 6.4) through nanoscopic intraparticle energy transfer.

Factors controlling fibroblast growth factor receptor-1's cytoplasmic trafficking and its regulation as revealed by FRAP analysis.

Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) "nuclear pathway," where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) "membrane pathway," in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1.

Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy.

A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.

Optical tracking of organically modified silica nanoparticles as DNA carriers: a nonviral, nanomedicine approach for gene delivery.

This article reports a multidisciplinary approach to produce fluorescently labeled organically modified silica nanoparticles as a nonviral vector for gene delivery and biophotonics methods to optically monitor intracellular trafficking and gene transfection. Highly monodispersed, stable aqueous suspensions of organically modified silica nanoparticles, encapsulating fluorescent dyes and surface functionalized by cationic-amino groups, are produced by micellar nanochemistry. Gel-electrophoresis studies reveal that the particles efficiently complex with DNA and protect it from enzymatic digestion of DNase 1. The electrostatic binding of DNA onto the surface of the nanoparticles, due to positively charged amino groups, is also shown by intercalating an appropriate dye into the DNA and observing the Forster (fluorescence) resonance energy transfer between the dye (energy donor) intercalated in DNA on the surface of nanoparticles and a second dye (energy acceptor) inside the nanoparticles. Imaging by fluorescence confocal microscopy shows that cells efficiently take up the nanoparticles in vitro in the cytoplasm, and the nanoparticles deliver DNA to the nucleus. The use of plasmid encoding enhanced GFP allowed us to demonstrate the process of gene transfection in cultured cells. Our work shows that the nanomedicine approach, with nanoparticles acting as a drug-delivery platform combining multiple optical and other types of probes, provides a promising direction for targeted therapy with enhanced efficacy as well as for real-time monitoring of drug action.

Ceramic-based nanoparticles entrapping water-insoluble photosensitizing anticancer drugs: a novel drug-carrier system for photodynamic therapy.

A novel nanoparticle-based drug carrier for photodynamic therapy is reported which can provide stable aqueous dispersion of hydrophobic photosensitizers, yet preserve the key step of photogeneration of singlet oxygen, necessary for photodynamic action. A multidisciplinary approach is utilized which involves (i) nanochemistry in micellar cavity to produce these carriers, (ii) spectroscopy to confirm singlet oxygen production, and (iii) in vitro studies using tumor cells to investigate drug-carrier uptake and destruction of cancer cells by photodynamic action. Ultrafine organically modified silica-based nanoparticles (diameter approximately 30 nm), entrapping water-insoluble photosensitizing anticancer drug 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide, have been synthesized in the nonpolar core of micelles by hydrolysis of triethoxyvinylsilane. The resulting drug-doped nanoparticles are spherical, highly monodispersed, and stable in aqueous system. The entrapped drug is more fluorescent in aqueous medium than the free drug, permitting use of fluorescence bioimaging studies. Irradiation of the photosensitizing drug entrapped in nanoparticles with light of suitable wavelength results in efficient generation of singlet oxygen, which is made possible by the inherent porosity of the nanoparticles. In vitro studies have demonstrated the active uptake of drug-doped nanoparticles into the cytosol of tumor cells. Significant damage to such impregnated tumor cells was observed upon irradiation with light of wavelength 650 nm. Thus, the potential of using ceramic-based nanoparticles as drug carriers for photodynamic therapy has been demonstrated.

A monomethine cyanine dye Cyan 40 for two-photon-excited fluorescence detection of nucleic acids and their visualization in live cells.

Monomethine cyanine dye 4-((1-methylbenzothiazolyliliden-2)methyl)-1,2,6-trimethylpyridinium perchlorate (Cyan 40) was investigated as a two-photon-excited fluorescence probe for nucleic acids (NA). Cyan 40 has been shown to demonstrate efficient two-photon-excited fluorescence in the presence of NA in vitro in contrast to solutions without NA. Two-photon confocal laser scanning microscopy (TPCLSM) and two-photon laser scanning microspectrofluorometry were used to check the possibility of using Cyan 40 as two-photon-excited fluorescence label for NA in living cells. Study of dye effect on viability of cells was also carried out. We ascertained that Cyan 40 is a cell-permeant dye, manifesting efficient two-photon-excited fluorescence when bound to NA in living cells, without any significant influence on viability of cells. TPCLSM images obtained from stained cells indicate preferential RNA staining by Cyan 40 compared with DNA.