Bimodal Lifetime Distribution Research Articles

Functionalizing Biodegradable Dextran Scaffolds Using Living Radical Polymerization: New Versatile Nanoparticles for the Delivery of Therapeutic Molecules

Conferring biodegradability to nanoparticles is vitally important when nanomedicine applications are being targeted, as this prevents potential problems with bioaccumulation of byproducts after delivery. In this work, dextran has been modified (and rendered hydrophobic) by partial acetalation. A solid state NMR method was first developed to fully characterize the acetalated polymers. In a subsequent synthetic step, RAFT functionality was attached via residual unmodified hydroxyl groups. The RAFT groups were then used in a living free radical polymerization reaction to control the growth of hydrophilic PEG-methacrylate chains, thereby generating amphiphilic comblike polymers. The amphiphilic polymers were then self-assembled in water to form various morphologies, including small vesicles, wormlike rods, and micellar structures, with PEG at the periphery acting as a nonfouling biocompatible polymer layer. The acetalated dextran nanoparticles were designed for potential doxorubicin (DOX) delivery application based on the premise that in the cell compartments (endosome, lysozome) the acetalated dextran would hydrolyze, destroying the nanoparticle structure, releasing the encapsulated DOX. In-vitro studies confirmed minimal cytotoxicity of the (unloaded) nanoparticles, even after 3 days, proving that the hydrolysis products from the acetal groups (methanol and acetone) had no observable cytotoxic effect. An intriguing initial result is reported that in vitro studies of DOX-loaded dextran-nanoparticles (compared to free DOX) revealed an increased differential toxicity toward a cancer cell line when compared to a normal cell line. Efficient accumulation of DOX in a human neuroblastoma cell line (SY-5Y) was confirmed by both confocal microscopy and flow cytometry measurements. Furthermore, the time dependent release of DOX was monitored using fluorescence lifetime imaging microscopy (FLIM) in SY-5Y live cells. FLIM revealed bimodal lifetime distributions, showing the accumulation of both DOX-loaded dextran-nanoparticles and subsequent release of DOX in the living cells. From FLIM data analysis, the amount of DOX released in SY-5Y cells was found to increase from 35% to 55% when the incubation time increased from 3 h to 24 h.

Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells

Frequency domain fluorescence lifetime imaging microscopy (FLIM) has been used in combination with laser scanning confocal microscopy to study the cellular uptake behavior of the antitumor drug doxorubicin (DOX) and micellar-encapsulated DOX (PLyAd-DOX). The endocytosis uptake process of PLyAd-DOX was monitored over 72 hours using confocal microscopy, with a maximum fluorescence recorded at incubation periods around 24 hours. The micellar structure was not found to release the encapsulated DOX during the time course of imaging. FLIM revealed single lifetime distributions of PLyAd-DOX during accumulation in the cytoplasm. The free DOX in contrast was observed both in the cytoplasm and the nuclear domain of the cell, showing bimodal lifetime distributions. There was a marked dependence of the measured free-DOX lifetime on concentration within the cell, in contrast to reference experiments in aqueous solution, where no such dependence was found. The results suggest the formation of macromolecular structures inside the living cells.

Perturbation of Conformational Dynamics of ASCUT-1 from Ascaris lumbricoides by Temperature and Sodium Dodecyl Sulfate.

ASCUT-1 is a protein found in cuticlin, the insoluble residue of the cuticles of the nematode Ascaris lumbricoides. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The monomeric form of ASCUT-1 contains a single tryptophan residue. An understanding of the structure-function relationship of the protein under different chemical-physical conditions is of fundamental importance for an understanding of its structure and function in cuticles. In this paper we report the effect of the temperature and sodium dodecyl sulfate on the structural stability of this protein. The structure of the protein was studied in the temperature range 25-85°C in the absence and in the presence of sodium dodecyl sulfate by frequency-domain measurements of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that the tryptophanyl emission decays were well described by a bimodal lifetime distribution, and that the temperature increases resulted in the sharpening and in the shortening of the tryptophanyl lifetime distribution. In the presence of SDS an unimodal fluorescence lifetime distribution as well as a marked decrease in the anisotropy decay values were observed.

Laurdan solvatochromism: solvent dielectric relaxation and intramolecular excited-state reaction

Absorption, steady-state, and time-resolved fluorescence measurements have been performed on laurdan dissolved either in white viscous apolar solvents or in ethanol as a function of temperature. The heterogeneity of the absorption spectra in white oils or in ethanol is consistent with semiempirical calculations performed previously on Prodan. From steady-state and time-resolved fluorescence measurements in apolar media, an excited state reaction is evidenced. The bimodal lifetime distribution determined from the maximum entropy method (MEM) analysis is attributed to the radiative deexcitation of a "locally excited" (LE) state and of a "charge transfer" (CT) state, whereas a very short component (20 ps), the sign and the amplitude of which depend on the emission wavelength, is attributed to the kinetics of the interconvertion reaction. The observation of an isoemissive point in the temperature range from -50 degrees C to -110 degrees C in ethanol suggests an interconvertion between two average excited-state populations: unrelaxed and solvent-relaxed CT states. A further decrease in temperature (-190 degrees C), leading to frozen ethanol, induces an additional and important blue shift. This low temperature spectrum is partly attributed to the radiative deexcitation of the LE state. Time-resolved emission spectra (TRES) measurements at -80 degrees C in the ethanol liquid phase show a large spectral shift of approximately 2500 cm(-1) (stabilization energy of the excited state: 7.1 kcal x M(-1)). The time-dependent fluorescence shift (TDFS) is described for its major part by a nanosecond time constant. The initial part of the spectral shift reveals, however, a subnanosecond process that can be due to fast internal solvent reorientation and/or to intramolecular excited-state reactions. These two relaxation times are also detected in the analysis of the fluorescence decays in the middle range of emission energy. The activation energy of the longest process is approximately 3 kcal x M(-1). At -190 degrees C, one subnanosecond and one nanosecond excited-state reactions are also evidenced. They are likely due to intramolecular rearrangements after the excitation, leading to the CT state and not to solvent relaxation, which is severely hindered in these temperature conditions. Therefore, both intramolecular and solvent relaxations are responsible for the large Stokes shift displayed by this probe as a function of solvent polarity. A possible scheme is proposed for the deexcitation pathway, taking into account the kinetics observed in these different solvents.

Perturbation of conformational dynamics, enzymatic activity, and thermostability of β-glycosidase from archaeonSulfolobus solfataricus by pH and sodium dodecyl sulfate detergent

The conformational dynamics of beta-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of beta-glycosidase similar to those observed in the absence of detergent.

Perturbation of conformational dynamics, enzymatic activity, and thermostability of β‐glycosidase from archaeon Sulfolobus solfataricus by pH and sodium dodecyl sulfate detergent

The conformational dynamics of β-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of β-glycosidase similar to those observed in the absence of detergent. Proteins 27:71–79 © 1997 Wiley-Liss, Inc.

Characterization of human immunodeficiency virus-1 (HIV-1) rev by (time-resolved) fluorescence spectroscopy

Fluorescence spectroscopy has been applied to the single tryptophan-containing regulatory protein Rev of human immunodeficiency virus (HIV-1). The fluorescence emission was found to have a maximum at 336 nm which refers to a surrounding of the chromophore of intermediate polarity. Fluorescence transients recorded at the maximum of fluorescence were found to decay nonexponentially. A bimodal lifetime distribution is obtained from exponential series analysis (ESM) with centers at 1.7 and 4.5 ns. Two microenvironments for tryptophan are suggested to be responsible for the two lifetime distributions. No innerfilter effect occurred in a Rev solution up to a concentration of 40 μM. A data quality study of ESM analysis as function of collected counts in the peak channel maximum (CIM) showed that, for reliable reconvolution, at least 15,000 CIM are necessary. The widths of the two distributions are shown to be temperature dependent. The broadening of the lifetime distributions when the temperature is raised to 50°C is interpreted as extension of the number of conformational substates which do not interconvert on the fluorescence time scale. The thermal deactivation (temperature quenching) is reflected in a constant decrease in the center of the short-lived lifetime distribution.

Unfolding Pathway of Apomyoglobin: Simultaneous Characterization of Acidic Conformational States by Frequency Domain Fluorometry

The dynamic properties of the conformational states co-existing during the acid-induced unfolding of tuna apomyoglobin, a single tryptophan-containing protein, have been investigated simultaneously by frequency domain fluorometry. In the transition region, in the absence of salt, the tryptophanyl fluorescence emission arises from a bimodal lifetime distribution. The pH decrease causes a marked broadening of the short-lived distribution component whereas the other component, i.e. the long-lived one, remains unchanged and represented by a very narrow lifetime distribution whose width is similar to that of the native protein. The broadening of the short-lived distribution component observed on lowering the pH indicated that this component arises from fully unfolded molecules. This was further corroborated by acrylamide quenching studies at acidic pH. The collisional quenching rate constant of the short-lived distribution component, i.e. 8·9 × 109 M-1 s-1, was found to be similar to that observed for a fully exposed residue. The long-lived distribution component was characterized by a lower collisional quenching rate constant, i.e.2·3 × 109 M-1 s-1. This value if compared to that determined for the native apoprotein at neutral pH, i.e. 4·0 × 108 M-1 s-1, indicates that the native-like structure surviving the acid-induced transition possesses a large molecular flexibility.

Time-resolved fluorescence studies of tomaymycin bonding to DNA.

Tomaymycin is an antibiotic that reacts at guanine N2 in the minor groove of the DNA helix. The number and type of tomaymycin-DNA adducts present on natural sequence DNA were identified using time-resolved fluorescence spectroscopy. At low bonding density, only two discrete species were observed with lifetimes of 4.3 and 7.1 ns and relative amplitudes of 40% and 60%. These two lifetime species are proposed to represent either R5' or S5' and S3' binding modes at the preferred bonding sequence 5'-AGA. R and S denote the configuration at C11 of tomaymycin, and 5' and 3' describe the orientation of the aromatic ring on the covalently modified strand. These two species were present over a range of solution conditions, including pH, nucleotide to drug ratio, DNA concentration, and DNA size. They have the same emission spectra, but slightly shifted absorption spectra. The weak temperature dependence of the fluorescence lifetimes presumably is due to the excited-state proton-transfer reaction that quenches tomaymycin fluorescence. The rate of formation of the longer lifetime species of DNA adduct is about twice as fast as that of the shorter lifetime species. Under saturating conditions, the fluorescence decay shows a bimodal lifetime distribution whether analyzed by least-squares assuming a Gaussian distribution model or by the maximum entropy method. The two groups of lifetimes are centered around 2-3 and 6-6.6 ns, reflecting multiple species on different bonding sequences.

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.

Bimodal lifetime distributions of dielectrics for integrated circuits

Constant voltage stressing of large area capacitors with thin thermal oxide and ONO dielectrics reveals that defect related breakdown is the dominant failure mode. Elimination of the complete early failure mode by means of screening will neither be possible nor necessary. Consequently, prediction of failure rates under operating conditions must be deduced from defect-related distributions. A statistical model is introduced and discussed in order to extrapolate to small failure percentages. Furthermore an additional term is introduced in the effective thickness model of Lee et al. which takes into account the weaker field acceleration behaviour of very thin dielectrics.

Fluorescence lifetime distributions of parinaroyl phospholipids in choline plasmalogen and phosphatidylcholine bilayers containing different amounts of cholesterol

The fluorescence decay of alkenylparinaroyl- and palmitoylparinaroyl glycerophosphocholines in vesicles of the unlabeled alkenyloleoyl and palmitoyloleoyl analogs was determined by multifrequency phase and modulation fluorometry. The measured phase angles and demodulations could be equally well fitted to a biexponential decay, as well as unimodal or bimodal continuous lifetime distributions. The latter model was applied to study the influence of cholesterol on parinaroyl phospholipid fluorescence in vesicles. The long-living component of a bimodal lifetime distribution was sensitive toward the presence of the sterol. Upon increasing cholesterol concentrations, its lifetime center increased and its distribution widths decreased. Lifetime distribution widths in vesicles of alkenyloleoyl- or palmitoyloleoyl-glycerophosphocholine (choline plasmalogen and phosphatidylcholine, respectively) were reduced by the sterol to the same extent. We interprete the sterol-induced lifetime distribution narrowing as an effect due to an increase of membrane homogeneity in cholesterol-phospholipid membranes.