Nanoparticles combining enhanced cellular drug delivery with efficient fluorescence detection are important tools for the development of theranostic agents. Here, we demonstrate this concept by a simple, fast, and robust protocol of cationic polymer-mediated gold nanocluster (Au NCs) self-assembly into nanoparticles (NPs) of ca. 120 nm diameter. An extensive characterization of the monodisperse and positively charged NPs revealed pH-dependent swelling properties, strong fluorescence enhancement, and excellent colloidal and photostability in water, buffer, and culture medium. The versatility of the preparation is demonstrated by using different Au NC surface ligands and cationic polymers. Steady-state and time-resolved fluorescence measurements give insight into the aggregation-induced emission phenomenon (AIE) by tuning the Au NC interactions in the self-assembled nanoparticles using the pH-dependent swelling. In vitro studies in human monocytic cells indicate strongly enhanced uptake of the NPs compared to free Au NCs in endocytic compartments. The NPs keep their assembly structure with quite low cytotoxicity up to 500 μg Au/mL. Enhanced drug delivery is demonstrated by loading peptides or antibodies in the NPs using a one-pot synthesis. Fluorescence microscopy and flow cytometry confirmed intracellular colocalization of the biomolecules and the NP carriers with a respective 1.7-fold and 6.5-fold enhanced cellular uptake of peptides and antibodies compared to the free biomolecules.

Significant improvements in the performances of the Super-ACO storage ring free-electron laser (FEL) at 800 MeV have been obtained recently: enhancement of the output power in the ultraviolet, laser duration of 10 h for the same injection of positrons, long-term stability with a micropulse of 60 ps FWHM. A first series of experiments using this FEL has then been successfully performed. Taking advantage of the time structure, the polarization and the high power of the source at 350 nm, the polarized fluorescence decays of the reduced nicotinamide adenine dinucleotide coenzyme (NADH) were studied in aqueous solution, using the single-photon counting (SPC) technique. The experimental setup is described, including the Super-ACO FEL characteristics and diagnostics. The FEL working point has been first optimized by monitoring the SPC apparatus function. A complete fluorescence experiment required about 30 min of data acquisition, during which the best integrated instrumental response had a FWHM of 110 ps. Measurements performed in such a way lead to the unambiguous separation of two close lifetime components of 0.28 and 0.62 ns in the fluorescence decays of NADH at 20 °C, in good agreement with previous works. The thermodynamic parameters obtained from temperature studies show that the NADH fluorescence heterogeneity is consistent with the ground-state folding equilibrium of the coenzyme, as characterized by many other spectroscopic techniques. From the fluorescence anisotropy decays, an apparent hydrodynamic radius of about 6 Å is determined, while on the other hand, a large initial depolarization of the fluorescence indicates a fast independent motion of the nicotinamide ring. The quality of the collected data fully meets the requirements for the study of more complex systems such as fluorescent compounds bound to proteins or membranes. Thus, the feasibility of use of a storage ring UV FEL for this type of time-resolved experiments on the subnanosecond time scale has been demonstrated.

We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.

Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33 % average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP-YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies.

The steady-state tryptophan fluorescence and time-resolved tryptophan fluorescence of Escherichia coli thioredoxin, calf thymus thioredoxin, and yeast thioredoxin have been studied. In all proteins, the tryptophan residues undergo strong static and dynamic quenching, probably due to charge-transfer interactions with the nearby sulfur atoms of the active cysteines. The use of a high-resolution photon counting instrument, with a time response of 60 ps full width at half-maximum, allowed the detection of fluorescence lifetimes ranging from a few tens of picoseconds to 10 ns. The data were analyzed both by classical nonlinear least squares and by a new method of entropy maximization (MEM) for the recovery of lifetime distributions. Simulations representative of the experimental data were used to test the MEM analysis. Strong support was obtained in this way for a small number of averaged discrete species in the fluorescence decays. Wavelength studies show that each of these components spreads over closely spaced excited states, while the temperature studies indicate that they do not exchange significantly on the nanosecond time scale. The oxidized form of thioredoxin is characterized by a high content of a very short lifetime below 70 ps, the amplitude of which is sharply decreased upon reduction. On the other hand, the fluorescence anisotropy decays indicate that reduction causes an increase of the very fast tryptophan rotations in an otherwise relatively rigid structure. While the calf thymus and E. coli proteins have mostly similar dynamical fluorescence properties, the yeast thioredoxin differs in many respects.

The binding of a £uorescent agonist to the acetycholine receptor from Torpedo electric organ has been studied by time-resolved spectroscopy in three di¡erent environments: in native membrane fragments, in the detergent CHAPS, and after complexation by amphipathic polymers ('amphipols'). Binding kinetics was similar in the membrane and in amphipols, demonstrating that the receptor can display unaltered allosteric transitions outside its natural lipid environment. In contrast, allosteric equilibria were strongly shifted towards the desensitized state in CHAPS. Therefore, the e¡ect of CHAPS likely results from molecular interactions rather than from the loss of bulk physical properties of the membrane environment. ß

The time-resolved fluorescence emission characteristics of the single tryptophan residue (Trp-59) of horse heart apocytochrome c--the precursor of the intramitochondrial cytochrome c--were studied in aqueous solution. The total fluorescence intensity decay measured over the whole emission spectrum was analyzed as a sum of three or four exponentials by the nonlinear least-squares method, the last model always providing a slight but significant decrease in the chi 2 values. Maximum entropy analysis, recently developed for time-resolved fluorometry (Livesey et al., 1987; Livesey & Brochon, 1987), strongly suggests the existence of a distribution including at least four separate classes of lifetimes. The center values were around 0.1-0.2, 1, 3, and 5 ns, in agreement with the lifetime values obtained by nonlinear least-squares regression analysis. As a function of the emission wavelength, these values remained constant within the experimental error, whereas a redistribution of the fractional amplitudes was observed: the contributions of the short components increased in the blue edge region of the emission spectrum. Temperature increase led essentially to a redistribution of the fractional amplitudes, affecting mostly that of the 5-ns component, which almost totally disappeared at high temperature (35-40 degrees C). The lifetime values were not significantly affected except for the 3-ns component, which decreased by about 15% in the temperature range studied. Such observations strongly suggest that the protein exists under different conformational substates in thermal equilibrium. Time-resolved fluorescence anisotropy measurements evidenced the existence of fast internal rotation of the Trp residue. An average maximum restricted angle of rotation of around 55 degrees was calculated. A second internal motion, slower by 1 order of magnitude, corresponding likely to a local motion of the peptide chain involving the Trp-59 residue, was detected on the anisotropy decay curve. Finally, the longest correlation time (5 ns) should correspond to the average rotation of the overall protein. Its value doubled as a function of the protein concentration, revealing an association process leading most likely to a dimer in the concentration range studied (2-139 microM). The flexibility of the peptide chain was more restrained in the associated than in the monomeric form, but the fast internal rotation of the Trp residue was not.

The fluorescence anisotropy decay of polybutadiene labeled with anthracene in the middle of the chain and embedded in a matrix of unlabeled polybutadiene is studied in the temperature range -50 to +80 °C. Very good precision is achieved thanks to the use of synchrotron radiation as the exciting source. The anisotropy is compared to different phenomenological expressions and molecular models proposed in the literature, using a multicriteria evaluation procedure. Intramolecular connectivity is shown to play an essential role in orientational dynamics in the melt. We also show that rather local orientational motions follow the same temperature law as macroscopic mechanical properties.