PD1 blockade potentiates the therapeutic efficacy of photothermally-activated and MRI-guided low temperature-sensitive magnetoliposomes.

This study investigates the effect of PD1 blockade on the therapeutic efficacy of novel doxorubicin-loaded temperature-sensitive liposomes. Herein, we report photothermally-activated, low temperature-sensitive magnetoliposomes (mLTSL) for efficient drug delivery and magnetic resonance imaging (MRI). The mLTSL were prepared by embedding small nitrodopamine palmitate (NDPM)-coated iron oxide nanoparticles (IO NPs) in the lipid bilayer of low temperature-sensitive liposomes (LTSL), using lipid film hydration and extrusion. Doxorubicin (DOX)-loaded mLTSL were characterized using dynamic light scattering, differential scanning calorimetry, electron microscopy, spectrofluorimetry, and atomic absorption spectroscopy. Photothermal experiments using 808 nm laser irradiation were conducted. In vitro photothermal DOX release studies and cytotoxicity was assessed using flow cytometry and resazurin viability assay, respectively. In vivo DOX release and tumor accumulation of mLTSL(DOX) were assessed using fluorescence and MR imaging, respectively. Finally, the therapeutic efficacy of PD1 blockade in combination with photothermally-activated mLTSL(DOX) in CT26-tumor model was evaluated by monitoring tumor growth, cytokine release and immune cell infiltration in the tumor tissue. Interestingly, efficient photothermal heating was obtained by varying the IO NPs content and the laser power, where on-demand burst DOX release was achievable in vitro and in vivo. Moreover, our mLTSL exhibited promising MR imaging properties with high transverse r2 relaxivity (333 mM-1 s-1), resulting in superior MR imaging in vivo. Furthermore, mLTSL(DOX) therapeutic efficacy was potentiated in combination with anti-PD1 mAb, resulting in a significant reduction in CT26 tumor growth via immune cell activation. Our study highlights the potential of combining PD1 blockade with mLTSL(DOX), where the latter could facilitate chemo/photothermal therapy and MRI-guided drug delivery.

A new twist in the photophysics of the GFP chromophore: a volume-conserving molecular torsion couple.

The simple structure of the chromophore of the green fluorescent protein (GFP), a phenol and an imidazolone ring linked by a methyne bridge, supports an exceptionally diverse range of excited state phenomena. Here we describe experimentally and theoretically the photochemistry of a novel sterically crowded nonplanar derivative of the GFP chromophore. It undergoes an excited state isomerization reaction accompanied by an exceptionally fast (sub 100 fs) excited state decay. The decay dynamics are essentially independent of solvent polarity and viscosity. Excited state structural dynamics are probed by high level quantum chemical calculations revealing that the fast decay is due to a conical intersection characterized by a twist of the rings and pyramidalization of the methyne bridge carbon. The intersection can be accessed without a barrier from the pre-twisted Franck-Condon structure, and the lack of viscosity dependence is due to the fact that the rings twist in the same direction, giving rise to a volume-conserving decay coordinate. Moreover, the rotation of the phenyl, methyl and imidazolone groups is coupled in the sterically crowded structure, with the methyl group translating the rotation of one ring to the next. As a consequence, the excited state dynamics can be viewed as a torsional couple, where the absorbed photon energy leads to conversion of the out-of-plane orientation from one ring to the other in a volume conserving fashion. A similar modification of the range of methyne dyes may provide a new family of devices for molecular machines, specifically torsional couples.

Ultrafast photophysics of the environment-sensitive 4'-methoxy-3-hydroxyflavone fluorescent dye.

The excited state intramolecular proton transfer (ESIPT) of 3-hydroxyflavone derivatives results in a fluorescence spectrum composed of two emission bands, the relative intensity of which is strongly influenced by the interaction with the local environment. We use time-resolved fluorescence and ultrafast transient absorption spectroscopies to investigate the photophysics of 4'-methoxy-3-hydroxyflavone in different solvents characterized by various polarities and hydrogen (H) bonding capabilities. We evidence that in this compound, the ESIPT reaction rate varies by more than 3 orders of magnitude, depending on the H-bonding capability of its local environment. This remarkable property is attributed to the moderate electron-donating strength of the 4'-methoxy substituent, and turns this fluorescent dye into a very promising fluorescent probe of biomolecular structures and interactions, where local structural heterogeneity may possibly be revealed by resolving a distribution of ESIPT reaction rates.

Ultrafast Dynamics in Light-Driven Molecular Rotary Motors Probed by Femtosecond Stimulated Raman Spectroscopy.

Photochemical isomerization in sterically crowded chiral alkenes is the driving force for molecular rotary motors in nanoscale machines. Here the excited-state dynamics and structural evolution of the prototypical light-driven rotary motor are followed on the ultrafast time scale by femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption (TA). TA reveals a sub-100-fs blue shift and decay of the Franck-Condon bright state arising from relaxation along the reactive potential energy surface. The decay is accompanied by coherently excited vibrational dynamics which survive the excited-state structural evolution. The ultrafast Franck-Condon bright state relaxes to a dark excited state, which FSRS reveals to have a rich spectrum compared to the electronic ground state, with the most intense Raman-active modes shifted to significantly lower wavenumber. This is discussed in terms of a reduced bond order of the central bridging bond and overall weakening of bonds in the dark state, which is supported by electronic structure calculations. The observed evolution in the FSRS spectrum is assigned to vibrational cooling accompanied by partitioning of the dark state between the product isomer and the original ground state. Formation of the product isomer is observed in real time by FSRS. It is formed vibrationally hot and cools over several picoseconds, completing the characterization of the light-driven half of the photocycle.

Ultrafast Excited State Dynamics in Molecular Motors: Coupling of Motor Length to Medium Viscosity.

Photochemically driven molecular motors convert the energy of incident radiation to intramolecular rotational motion. The motor molecules considered here execute four step unidirectional rotational motion. This comprises a pair of successive light induced isomerizations to a metastable state followed by thermal helix inversions. The internal rotation of a large molecular unit required in these steps is expected to be sensitive to both the viscosity of the medium and the volume of the rotating unit. In this work, we describe a study of motor motion in both ground and excited states as a function of the size of the rotating units. The excited state decay is ultrafast, highly non-single exponential, and is best described by a sum of three exponential relaxation components. The average excited state decay time observed for a series of motors with substituents of increasing volume was determined. While substitution does affect the lifetime, the size of the substituent has only a minor effect. The solvent polarity dependence is also slight, but there is a significant solvent viscosity effect. Increasing the viscosity has no effect on the fastest of the three decay components, but it does lengthen the two slower decay times, consistent with them being associated with motion along an intramolecular coordinate displacing a large solvent volume. However, these slower relaxation times are again not a function of the size of the substituent. We conclude that excited state decay arises from motion along a coordinate which does not necessarily require complete rotation of the substituents through the solvent, but is instead more localized in the core structure of the motor. The decay of the metastable state to the ground state through a helix inversion occurs 14 orders of magnitude more slowly than the excited state decay, and was measured as a function of substituent size, solvent viscosity and temperature. In this case neither substituent size nor solvent viscosity influences the rate, which is entirely determined by the activation barrier. This result is different to similar studies of an earlier generation of molecular motors, which suggests different microscopic mechanisms are in operation in the different generations. Finally, the rate of photochemical isomerization was studied for the series of motors, and those with the largest volume substituents showed the highest photochemical cross section.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.

Ultrafast excited state dynamics in 9,9'-bifluorenylidene.

9,9'-Bifluorenylidene has been proposed as an alternative and flexible electron acceptor in organic photovoltaic cells. Here we characterize its excited state properties and photokinetics, combining ultrafast fluorescence and transient IR measurements with quantum chemical calculations. The fluorescence decay is ultrafast (sub-100 fs) and remarkably independent of viscosity. This suggests that large scale structure change is not the primary relaxation mode. The ultrafast decay populates a dark state characterized by distinct vibrational and electronic spectra. This state decays with a 6 ps time constant to a hot ground state that ultimately populates the initial state with a 20 ps time constant; these times are also insensitive to solvent viscosity. No metastable intermediate structures are resolved in the photocycle after population of the dark state. The implications of these results for the operation of 9,9'-bifluorenylidene as an electron acceptor and as a potential molecular switch are discussed.

Chemically optimizing operational efficiency of molecular rotary motors.

Unidirectional molecular rotary motors that harness photoinduced cis-trans (E-Z) isomerization are promising tools for the conversion of light energy to mechanical motion in nanoscale molecular machines. Considerable progress has been made in optimizing the frequency of ground-state rotation, but less attention has been focused on excited-state processes. Here the excited-state dynamics of a molecular motor with electron donor and acceptor substituents located to modify the excited-state reaction coordinate, without altering its stereochemistry, are studied. The substituents are shown to modify the photochemical yield of the isomerization without altering the motor frequency. By combining 50 fs resolution time-resolved fluorescence with ultrafast transient absorption spectroscopy the underlying excited-state dynamics are characterized. The Franck-Condon excited state relaxes in a few hundred femtoseconds to populate a lower energy dark state by a pathway that utilizes a volume conserving structural change. This is assigned to pyramidalization at a carbon atom of the isomerizing bridging double bond. The structure and energy of the dark state thus reached are a function of the substituent, with electron-withdrawing groups yielding a lower energy longer lived dark state. The dark state is coupled to the Franck-Condon state and decays on a picosecond time scale via a coordinate that is sensitive to solvent friction, such as rotation about the bridging bond. Neither subpicosecond nor picosecond dynamics are sensitive to solvent polarity, suggesting that intramolecular charge transfer and solvation are not key driving forces for the rate of the reaction. Instead steric factors and medium friction determine the reaction pathway, with the sterically remote substitution primarily influencing the energetics. Thus, these data indicate a chemical method of optimizing the efficiency of operation of these molecular motors without modifying their overall rotational frequency.

Ultrafast excited state dynamics of the green fluorescent protein chromophore and its kindling fluorescent protein analogue.

Fluorescent proteins exhibit a very diverse range of photochemical behaviour, from efficient fluorescence through photochromism to photochemical reactivity. Remarkably this diverse behaviour arises from chromophores which have very similar structures. Here we describe measurements and modelling of the excited state dynamics in the chromophores of GFP (HBDI) and the kindling fluorescent protein, KFP (FHBMI). The methods are ultrafast fluorescence spectroscopy with sub 50 fs time resolution and the modelling is based on the Smoluchowski equation. The excited state decays of both chromophores are very fast, longer for their anions than for the neutral form and independent of wavelength. Detailed studies show the mean fluorescence wavelength to be independent of time. The excited state decay times are also observed to be a very weak function of solvent polarity and viscosity. These results are modelled utilising recently calculated potential energy surfaces for the ground and excited states as a function of the twist coordinates about the two bridging bonds of the chromophore. For FHBMI and the scarce data on the neutral HBDI the calculations are not successful suggesting the need for refinement of these potential energy surfaces. For HBDI in methanol the simulation is successful provided a strong dependence of the radiationless decay rate on the coordinate is assumed. Such dependence should be included in future calculations of excited state dynamics. When the simulations are extended to more viscous solvents they fail to reproduce the observed weak viscosity dependence. The implications of these results for the nature of the coordinate leading to radiationless decay in the chromophore and for the photodynamics of fluorescent proteins are discussed.

Ultrafast dynamics in the power stroke of a molecular rotary motor.

Light-driven molecular motors convert light into mechanical energy through excited-state reactions. Unidirectional rotary molecular motors based on chiral overcrowded alkenes operate through consecutive photochemical and thermal steps. The thermal (helix inverting) step has been optimized successfully through variations in molecular structure, but much less is known about the photochemical step, which provides power to the motor. Ultimately, controlling the efficiency of molecular motors requires a detailed picture of the molecular dynamics on the excited-state potential energy surface. Here, we characterize the primary events that follow photon absorption by a unidirectional molecular motor using ultrafast fluorescence up-conversion measurements with sub 50 fs time resolution. We observe an extraordinarily fast initial relaxation out of the Franck-Condon region that suggests a barrierless reaction coordinate. This fast molecular motion is shown to be accompanied by the excitation of coherent excited-state structural motion. The implications of these observations for manipulating motor efficiency are discussed.

Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein Chromophore.

Cis-trans photoisomerization is proposed as a key process in the photoswitching of some photoactivatable fluorescent proteins. Here we present ultrafast fluorescence measurements of the model GFP chromophore (HBDI) in the cis state and in a mixture of the cis and trans states. Our results demonstrate that the mean lifetimes of the cis and trans states are remarkably similar. Therefore, the specific isomer of the chromophore cannot be solely responsible for the different photophysics of the bright and dark states of photoactive proteins, which must therefore be due to differential interactions between the different isomers of the chromophore and the protein.

Chemically modulating the photophysics of the GFP chromophore.

There is growing interest in engineering the properties of fluorescent proteins through modifications to the chromophore structure utilizing mutagenesis with either natural or unnatural amino acids. This entails an understanding of the photophysical and photochemical properties of the modified chromophore. In this work, a range of GFP chromophores with different alkyl substituents are synthesized and their electronic spectra, pH dependence, and ultrafast fluorescence decay kinetics are investigated. The weakly electron donating character of the alkyl substituents leads to dramatic red shifts in the electronic spectra of the anions, which are accompanied by increased fluorescence decay times. This high sensitivity of electronic structure to substitution is also characteristic of some fluorescent proteins. The solvent viscosity dependence of the decay kinetics are investigated, and found to be consistent with a bimodal radiationless relaxation coordinate. Some substituents are shown to distort the planar structure of the chromophore, which results in a blue shift in the electronic spectra and a strong enhancement of the radiationless decay. The significance of these data for the rational design of novel fluorescent proteins is discussed.

Reactive dynamics in confined liquids: interfacial charge effects on ultrafast torsional dynamics in water nanodroplets.

The excited-state dynamics of a reactive dye molecule, auramine O, have been studied in nanoscale water droplets stabilized by a nonionic surfactant. Spectral dynamics were measured as a function of the radius of the water nanodroplet with 50 fs time resolution using time-resolved fluorescence up-conversion method. Qualitatively, the effect of confinement is to dramatically slow the rate of the reaction compared to that of bulk water. Data were quantitatively analyzed using the one-dimensional generalized Smoluchowski equation assuming a time-dependent diffusion coefficient. The results were contrasted with our earlier analysis of auramine O in aqueous nanodroplets stabilized by the ionic surfactant AOT. The excited-state reaction is slower in the nonionic surfactant, showing that interfacial charge is not required to suppress reactions in nanoscale water droplets. The location of the dye in the heterogeneous micelle is investigated by comparing the absorption spectra of AO in the micelle with those of a water- polyethyleneglycol mixture (to mimic the surfactant head group). The results suggest that the charged dye is located in the water phase.