Uniform silica nanoparticles encapsulating two-photon absorbing fluorescent dye

We have prepared uniform silica nanoparticles (NPs) doped with a two-photon absorbing zwitterionic hemicyanine dye by reverse microemulsion method. Obvious solvatochromism on the absorption spectra of dye-doped NPs indicates that solvents can partly penetrate into the silica matrix and then affect the ground and excited state of dye molecules. For dye-doped NP suspensions, both one-photon and two-photon excited fluorescence are much stronger and recorded at shorter wavelength compared to those of free dye solutions with comparative overall dye concentration. This behavior is possibly attributed to the restricted twisted intramolecular charge transfer (TICT), which reduces fluorescence quenching when dye molecules are trapped in the silica matrix. Images from two-photon laser scanning fluorescence microscopy demonstrate that the dye-doped silica NPs can be actively uptaken by Hela cells with low cytotoxicity.     

Fluorescent AIE dots encapsulated organically modified silica (ORMOSIL) nanoparticles for two-photon cellular imaging

2,3-Bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl) fumaronitrile (TPE-TPA-FN or TTF), which possesses aggregation-induced emission (AIE) characteristic, is doped in organically modified silica (ORMOSIL) nanoparticles. By increasing the weight ratio of TTF to the precursor of silica nanoparticles (the quantities of the precursors were kept the same), the fluorescence intensity of nanoparticles increased correspondingly, due to the formation of larger AIE dots in the cores of ORMOSIL nanoparticles. The fluorescent and biocompatible nanoprobes were then utilized for in vitro imaging of HeLa cells. Two-photon fluorescence microscopy clearly illustrated that the nanoparticles have the capacity of nucleus permeability, as well as cytoplasm staining towards tumor cells. Our experimental results may offer a promising method for fast and bright fluorescence imaging, as well as bio-molecule/drug delivery to cell nucleus.     

Surface functionalization of two-photon dye-doped mesoporous silica nanoparticles with folic acid: cytotoxicity studies with HeLa and MCF-7 cancer cells

Two-photon dye-doped mesoporous silica nanoparticles (NPs) have been conjugated with folic acid (FA) in order to obtain efficient nanotools for bioimaging of cancer cells. The surface of the NPs was first functionalized with 3-aminopropyltriethoxysilane. The amine-covered NPs were subsequently reacted with an activated ester derivative of FA. Cytotoxicity studies performed with MCF7 and HeLa cancer cells demonstrate that these functionalized NPs are much less cytotoxic than the non-functionalized NPs against both cell lines. Unfortunately, the grafting of FA enables the formation of charge transfer complexes between the two-photon dye and FA which leads to quenching of the fluorescence of the NPs. Hence although these NPs cannot be used for biomaging purposes, they offer interesting potentialities if the two-photon dye used can be replaced by a two-photon fluorophore which do not interact with FA or if the interaction between the encapsulated dye and FA can be prevented by using a suitable spacer between the surface and the FA moiety.     

Fluorescence resonance energy transfer in a novel two-photon absorbing system

A novel fluorescence resonance energy transfer (FRET) system containing a two-photon absorbing dye and a nile red chromophore has been synthesized. Upon two-photon excitation by laser at 815 nm this molecule displays efficient energy transfer from the two-photon absorbing dye to the nile red moiety, with an 8-fold increase in emission compared to the model compound. Similarly, single-photon excitation of the two-photon absorbing moiety at 405 nm results in >99% energy-transfer efficiency, along with a 3.4-fold increase in nile red emission compared to direct excitation of the nile red chromophore at 540 nm. This system provides an effective way to use IR radiation to excite molecules that, by themselves, have little or no two-photon absorption.     

Reversible two-photon photoswitching and two-photon imaging of immunofunctionalized nanoparticles targeted to cancer cells

Both photoswitchable fluorescent nanoparticles and photoactivatable fluorescent proteins have been used for super-resolution far-field imaging on the nanometer scale, but the photoactivating wavelength for such photochemical events generally falls in the near-UV (NUV) region (<420 nm), which is not preferred in cellular imaging. However, using two near-IR (NIR) photons that are lower in energy, we can circumvent such problems and replace NUV single-photon excitations (e.g., 390 nm) with NIR two-photon excitations (e.g., 780 nm). Thus, we have demonstrated that alternating 780 nm NIR two-photon and 488 nm single-photon excitations induces reversible on-off fluorescence switching of immunotargeted nanoparticles in the human breast cancer cell line SK-BR-3. Herein, two-photon absorption not only caused spiropyran-merocyanine photoisomerization within the particles but also imparted red fluorescence. In comparison with single-photon NUV excitations, two-photon NIR laser excitations can potentially reduce absorption-related photodamage to living systems because cellular systems absorb much more weakly in the NIR.     

Enhanced two-photon singlet oxygen generation by photosensitizer-doped conjugated polymer nanoparticles

We have prepared photosensitizer-doped conjugated polymer nanoparticles by using a reprecipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP). These TPP-doped PFEMO nanoparticles are stable and have a uniform size of ～50 nm. Efficient intraparticle energy transfer from PFEMO to TPP has been observed. The TPP emission of the nanoparticles was found to be enhanced by 21-fold by PFEMO under two-photon excitation. Enhanced two-photon excitation singlet oxygen generation efficiency in the TPP-doped PFEMO nanoparticles has been demonstrated. Our results suggest that these photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.     

双重对象同时检测的新型荧光纳米粒子传感器的制备

报道了一种基于荧光共振能量转移理论合成的新型纳米粒子. 新型纳米粒子A中掺杂了由亲和素-生物素桥联并可发生荧光共振能量转移(FRET)的供体染料和受体染料; 新型纳米粒子B中只掺杂了供体染料. 在使用供体染料的特征激发波长同时激发纳米粒子A和B时, 纳米粒子A由于荧光共振能量转移而发射出受体染料的特征波长, 而纳米粒子B则只发射供体染料的特征波长. 这样, 在单一波长激发下, 可很容易地实现双重对象同时检测.     

吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒的制备及其性质研究

以蛋白质或多肽修饰的吲哚类菁染料Cy3为内核, 采用实验条件简单的油包水反相微乳液方法成核, 通过正硅酸乙酯水解形成的网状二氧化硅包壳的方法制备吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒. 考察了以不同等电点的蛋白质和多肽修饰的Cy3为内核材料对吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒制备的影响. 结果表明, 分别采用人免疫球蛋白(IgG)或多聚赖氨酸修饰的Cy3为内核材料, 都能制备荧光强度高、荧光稳定性强和染料泄漏极少的Cy3嵌入的核壳荧光纳米颗粒. 进一步对Cy3嵌入的核壳荧光纳米颗粒进行了表征, 并将基于这一新型的荧光纳米颗粒建立起来的生物标记方法初步应用于流感病毒DNA的检测, 其检测线性范围为3.18×10-10～1.27×10-9 mol/L, 检测下限为3.51×10-10 mol/L, 相关系数r为0.986 5.     

Hypocrellin B doped and pH-responsive silica nanoparticles for photodynamic therapy

pH-responsive ¹O₂ photosensitizing systems may serve as selective photodynamic therapy (PDT) agents by targeting the acidic interstitial fluid of many kinds of tumors. In this work, a natural and clinically used photosensitizer (Hypocrellin B, HB) and a pH indicator (Bromocresol Purple, BCP) were co-encapsulated in organically modified silica nanoparticles. BCP successfully regulated the ¹O₂ generation efficiency of HB through the “inner filter” effect, which shows much stronger ¹O₂ generation ability in an acidic than in a basic environment. In vitro experiments also demonstrated that HB-doped nanoparticles are effective in killing tumor cells by PDT.

     

Photodynamic action of Rose Bengal silica nanoparticle complex on breast and oral cancer cell lines

Rose Bengal, an anionic photosensitizer was conjugated to organically modified silica nanoparticles having 3-amino propyl groups by electrostatic or covalent interaction. The drug-nanoparticle complexes were characterized by FTIR, light scattering and zeta potential measurements. Significant changes were observed in the spectroscopic properties of the drug when it is conjugated with nanoparticles. The toxicity of the free drug and drug-nanoparticle complex was studied against oral (4451) and breast (MCF-7) cancer cell lines. Both complexes with nanoparticles were more phototoxic than free Rose Bengal, with the covalent complex being the more effective. Studies carried out on cellular uptake, photostability and singlet oxygen generation suggest that enhanced phototoxicity is primarily due to the enhanced uptake of the drug-nanoparticle complex.     

Preparation and characterization of poly(lipid)-coated, fluorophore-doped silica nanoparticles for biolabeling and cellular imaging

The fabrication, characterization, and implementation of poly(lipid)-coated, highly luminescent silica nanoparticles as fluorescent probes for labeling of cultured cells are described. The core of the probe is a sol-gel-derived silica nanoparticle, 65-100 nm in diameter, in which up to several thousand dye molecules are encapsulated (Lian, W.; et al. Anal. Biochem. 2004, 334, 135-144). The core is coated with a membrane composed of bis-sorbylphosphatidylcholine, a synthetic polymerizable lipid that is chemically cross-linked to enhance the environmental and chemical stability of the membrane relative to a fluid lipid membrane. The poly(lipid) coating has two major functions: (i) to reduce nonspecific interactions, based on the inherently biocompatible properties of the phosphorylcholine headgroup, and (ii) to permit functionalization of the particle, by doping the coating with lipids bearing chemically reactive or bioactive headgroups. Both functions are demonstrated: (i) Nonspecific adsorption of dissolved proteins to bare silica nanoparticles and of bare nanoparticles to cultured cells is significantly reduced by application of the poly(lipid) coating. (ii) Functionalization of poly(lipid)-coated nanoparticles with a biotin-conjugated lipid creates a probe that can be used to target both dissolved protein receptors as well as receptors on the membranes of cultured cells. Measurements performed on single nanoparticles bound to planar supported lipid bilayers verify that the emission intensity of these probes is significantly greater than that of single protein molecules labeled with several fluorophores.     

Eu3+,Tb3+掺杂荧光二氧化硅纳米粒子的合成及其结构、成分和荧光性质的调控

针对目前存在的稀土螯合物掺杂量低、发射吸收波长受到限制等问题,首先用设计好的敏化剂-配体-硅烷偶联剂来螯合稀土离子得到稀土螯合物,然后在反相微乳液中将上述稀土螯合物与正硅酸乙酯(TEOS)共同水解缩聚,合成了稀土掺杂的二氧化硅纳米粒子。通过这种方法,不仅实现了对FSNPs粒径、成分、发射波长的调控,还通过使用不同生色团和稀土离子,获得了对激发波长的调控能力。此外,进一步阐述了稀土掺杂FSNPs中的能量转移过程,提出了一种通过能级调节FSNPs荧光的新方法。     

Cell penetrating silica nanoparticles doped with two-photon absorbing fluorophores

Organosilica nanoparticles, doped with two-photon absorbing distyrylbenzene derivatives, were prepared and studied as cell staining agents. Two dyes were used, bearing either two peripheral dimethylamino groups or one dimethylamino and one cyano group. Due to the internal charge transfer character of their excited state, the dyes employed show a red-shifted quenched emission in polar solvents. Once included in the particles, the properties of the two dyes undergo a substantial variation. Particles doped with the cyano substituted distyrylbenzene show a remarkable emission quantum yield in water, probably due to solvent exclusion from the nanoparticle core. To the contrary, the emission of the particles containing the dye substituted with two dimethylamino groups is substantially quenched. Fluorescence emission induced by two-photon absorption follows the same behaviour. The doped nanoparticles can be rapidly internalized by tumour cells with accumulation limited to the cytoplasm and show no cytotoxicity at low concentrations.     

Excitation Energy Transfer from Semi‐Conducting Polymer Nanoparticles to Surface‐Bound Fluorescent Dyes

Summary: The first examples of the dye‐coated semi‐conducting polymer nanoparticles as well as experiments to demonstrate the excitation energy transfer from the excited chromophor of the nanoparticle to the fluorescent dye are described. We have demonstrated that the blue fluorescence of the dye‐coated polyfluorene nanoparticles is only slightly quenched after dye deposition. However, a new emission band of the surface‐bound dye (Rhodamine 6G or Rhodamine TM) appears in the wavelength region of 530–600 nm. These results clearly indicate an effective excitation energy transfer from the excited PF chromophores to the fluorescent dye.

Emission spectra of PF2/6 nanoparticle dispersion and of Rhodamine 6G‐coated nanoparticle dispersion.     

Photodynamic characterization and in vitro application of methylene blue-containing nanoparticle platforms

This article presents the development and characterization of nanoparticles loaded with methylene blue (MB), which are designed to be administered to tumor cells externally and deliver singlet oxygen (1O2) for photodynamic therapy (PDT), i.e. cell kill via oxidative stress to the membrane. We demonstrated the encapsulation of MB, a photosensitizer (PS), in three types of sub-200 nm nanoparticles, composed of polyacrylamide, sol-gel silica and organically modified silicate (ORMOSIL), respectively. Induced by light irradiation, the entrapped MB generated 1O2, and the produced 1O2 was measured quantitatively with anthracene-9,10-dipropionic acid, disodium salt, to compare the effects of different matrices on 1O2 delivery. Among these three different kinds of nanoparticles, the polyacrylamide nanoparticles showed the most efficient delivery of 1O2, but its loading of MB was low. In contrast, the sol-gel nanoparticles had the best MB loading but the least efficient 1O2 delivery. In addition to investigating the matrix effects, a preliminary in vitro PDT study using the MB-loaded polyacrylamide nanoparticles was conducted on rat C6 glioma tumor cells with positive photodynamic results. The encapsulation of MB in nanoparticles should diminish the interaction of this PS with the biological milieu, thus facilitating its systemic administration. Furthermore, the concept of the drug-delivering nanoparticles has been extended to a new type of dynamic nanoplatform (DNP) that only delivers 1O2. This DNP could also be used as a targeted multifunctional platform for combined diagnostics and therapy of cancer.     

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.     

Quantitative in vitro demonstration of two-photon photodynamic therapy using photofrin and visudyne

Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne and Photofrin are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed two-photon photosensitizers before proceeding to in vivo studies in preclinical animal models.     

Enhancement of two-photon absorption cross section and singlet-oxygen generation in porphyrin-cored star polymers

We report a newly synthesized polymer of a star-shaped porphyrin compound(TPA-FxP) with four oligofluorene arms at its meso positions with the pronounced enhancement of the two-photon properties and the generation of singlet oxygen by utilizing the two-photon excited fluorescence resonance energy transfer.The steady-state spectra and transient triplet-triplet absorption spectra give evidence that the enhanced two-photon absorption cross section results from not only the through-space energy transfer(Frster...     

The role of exciton hopping and direct energy transfer in the efficient quenching of conjugated polyelectrolytes

The dynamics of fluorescence quenching of a conjugated polyelectrolyte by a cyanine dye are investigated by femtosecond fluorescence up-conversion and polarization resolved transient absorption. The data are analyzed with a model based on the random walk of the exciton within the polymer chain and a long-range direct energy transfer between polymer and dye. We find that rapid intrachain energy migration toward complex sites with the dye leads to the highly efficient energy transfer, whereas the contribution from direct, long-range energy transfer is negligible. We determine the actual density of complexes with the dye along the polymer chain. A clear deviation from calculations based on a constant complex association constant is found and explained by a reduced effective polymer concentration due to aggregation. Altogether, the quenching efficiency is found to be limited by (i) the energetic disorder within the polymer chain and (ii) the formation of loose polymer aggregates.     

Preparation of Fluorescence Tunable Polymer Nanoparticles by One-step Mini-emulsion

Fluorescence tunable polymer nanoparticles were prepared by incorporating two hydrophobic fluorescent dyes (9, 10-diphenylanthracene: DPA and nitrobenzoxadiazolyl: NBD) into polymethylmethacrylate (PMMA) nanoparticles via one-step mini-emulsion polymerization method. The prepared fluorescent nanoparticles exhibit the spectral properties of both DPA and NBD dye, indicating that the two fluorophores have been incorporated into the nanoparticles. The nanoparticles greatly enhance the fluorescence emission of the two hydrophobic dyes in aqueous media probably by providing good protection of the dye molecules in the polymer nanoparticles matrix. Moreover, by varying the doping ratio of the two hydrophobic dyes, the polymer nanoparticles exhibit tunable and distinguishable emission characteristics under a single wavelength excitation via occuring fluorescence resonance energy transfer (FRET).