Tuning the Photophysical Properties of Aza-BODIPYs in the Near-Infrared Region by Introducing Electron-Donating Thiophene Substituents.

This study presents the synthesis, the spectroscopic and electrochemical properties of new bis- and tetra-substituted azaboron-dipyrromethene (aza-BODIPY) dyes substituted by different electron donating groups connected to the aza-BODIPY core through a thiophene unit. In line with theoretical calculations, experimental measurements point out the positive impact of the thiophene group that behave as a secondary donor group leading to an enhancement of the intramolecular charge transfer process in comparison to previously reported aza-BODIPY dyes. This heterocycle has also been found to tune the oxidative potential and to stabilize the electro-generated species.

Domes and semi-capsules as model systems for infrared microspectroscopy of biological cells.

It is well known that infrared microscopy of micrometer sized samples suffers from strong scattering distortions, attributed to Mie scattering. The state-of-the-art preprocessing technique for modelling and removing Mie scattering features from infrared absorbance spectra of biological samples is built on a meta model for perfect spheres. However, non-spherical cell shapes are the norm rather than the exception, and it is therefore highly relevant to evaluate the validity of this preprocessing technique for deformed spherical systems. Addressing these cases, we investigate both numerically and experimentally the absorbance spectra of 3D-printed individual domes, rows of up to five domes, two domes with varying distance, and semi-capsules of varying lengths as model systems of deformed individual cells and small cell clusters. We find that coupling effects between individual domes are small, corroborating previous related literature results for spheres. Further, we point out and illustrate with examples that, while optical reciprocity guarantees the same extinction efficiency for top vs. bottom illumination, a scatterer's internal field may be vastly different in these two situations. Finally, we demonstrate that the ME-EMSC model for preprocessing infrared spectra from spherical biological systems is valid also for deformed spherical systems.

Assembly of Aggregation-Induced Emission Active Bola-Amphiphilic Macromolecules into Luminescent Nanoparticles Optimized for Two-Photon Microscopy In Vivo.

The (Z) and (E)-isomers of an extended tetraphenylethylene-based chromophore with optimized two-photon-induced luminescence properties are separated and functionalized with water-solubilizing pendant polymer groups, promoting their self-assembly in physiological media in the form of small, colloidal stable organic nanoparticles. The two resulting fluorescent suspensions are then evaluated as potential two-photon luminescent contrast agents for intravital epifluorescence and two-photon fluorescence microscopy. Comparisons with previously reported works involving similar fluorophores devoid of polymer side chains illustrate the benefits of later functionalization regarding the control of the self-assembly of the nano-objects and ultimately their biocompatibility toward the imaged organism.

Deprotonation Dynamics of Guanine Radical Cations.

This review is dedicated to guanine radical cations (G+ )· that are precursors to oxidatively generated damage to DNA. (G+ )· are unstable in neutral aqueous solution and tend to lose a proton. The deprotonation process has been studied by time-resolved absorption experiments in which (G+ )· radicals are produced either by an electron abstraction reaction, using an external oxidant, or by low-energy/low-intensity photoionization of DNA. Both the position of the released proton and the dynamics of the process depend on the secondary DNA structure. While deprotonation in duplex DNA leads to (G-H1)· radicals, in guanine quadruplexes the (G-H2)· analogs are observed. Deprotonation in monomeric guanosine proceeds with a time constant of ~60 ns; in genomic DNA, it is completed within 2 µs; and in guanine quadruplexes, it spans from at least 30 ns to over 50 µs. Such a deprotonation dynamics in four-stranded structures, extended over more than three decades of times, is correlated with the anisotropic structure of DNA and the mobility of its hydration shell. In this case, commonly used second-order reaction models are inappropriate for its description.

3D Printing and Pyrolysis of Optical ZrO2 Nanostructures by Two-Photon Lithography: Reduced Shrinkage and Crystallization Mediated by Nanoparticles Seeds.

Two-photon lithography is a potential route to produce high-resolution 3D ceramics. However, the large shrinkage due to the elimination of an important organic counterpart of the printed material during debinding/sintering remains a lock to further development of this technology. To limit this phenomenon, an original approach based on a composite resin incorporating 45 wt% ultrasmall (5 nm) zirconia stabilized nanoparticles into the zirconium acrylate precursor is proposed to process 3D zirconia microlattices and nanostructured optical surfaces. Interestingly, the nanoparticles are used both as seeds allowing control of the crystallographic phase formed during the calcination process and as structural stabilizing agent preventing important shrinkage of the printed ceramic. After 3D photolithography and pyrolysis, the weight and volume loss of the microstructures are drastically reduced as compared to similar systems processed with the reference resin without nanoparticles, and stable 3D microstructures of cubic zirconia are obtained with high spatial resolution. In the case of a patterned surface, the refractive index of 2.1 leads to a diffraction efficiency large enough to obtain microfocusing with linewidths of 0.1 µm, and the demonstration of a microlens array with a period as small as 0.8 µm.

Hybrid multimodal contrast agent for multiscale in vivo investigation of neuroinflammation.

Neuroinflammation is a process common to several brain pathologies. Despites its medical relevance, it still remains poorly understood; there is therefore a need to develop new in vivo preclinical imaging strategies to monitor inflammatory processes longitudinally. We here present the development of a hybrid imaging nanoprobe named NP3, that was specifically designed to get internalized by phagocytic cells and imaged in vivo with MRI and bi-photon microscopy. NP3 is composed of a 16 nm core of gadolinium fluoride (GdF3), coated with bisphosphonate polyethylene glycol (PEG) and functionalized with a Lemke-type fluorophore. It has a hydrodynamic diameter of 28 ± 8 nm and a zeta potential of -42 ± 6 mV. The MR relaxivity ratio at 7 T is r1/r2 = 20; therefore, NP3 is well suited as a T2/T2* contrast agent. In vitro cytotoxicity assessments performed on four human cell lines revealed no toxic effects of NP3. In addition, NP3 is internalized by macrophages in vitro without inducing inflammation or cytotoxicity. In vivo, uptake of NP3 has been observed in the spleen and the liver. NP3 has a prolonged vascular remanence, which is an advantage for macrophage uptake in vivo. The proof-of-concept that NP3 may be used as a contrast agent targeting phagocytic cells is provided in an animal model of ischemic stroke in transgenic CX3CR1-GFP/+ mice using three complementary imaging modalities: MRI, intravital two-photon microscopy and phase contrast imaging with synchrotron X-rays. In summary, NP3 is a promising preclinical tool for the multiscale and multimodal investigation of neuroinflammation.

Two-Photon Absorbing AIEgens: Influence of Stereoconfiguration on Their Crystallinity and Spectroscopic Properties and Applications in Bioimaging.

This paper aims at designing chromophores with efficient aggregation-induced emission (AIE) properties for two-photon fluorescence microscopy (2PFM), which is one of the best-suited types of microscopy for the imaging of living organisms or thick biological tissues. Tetraphenylethylene (TPE) derivatives are common building blocks in the design of chromophores with efficient AIE properties. Therefore, in this study, extended TPE AIEgens specifically optimized for two-photon absorption (2PA) are synthesized and the resulting (E/Z) isomers are separated using chromatography on chiral supports. Comparative characterization of the AIE properties is performed on the pure (Z) and (E) isomers and the mixture, allowing us, in combination with powder X-ray diffraction and solid-state NMR, to document a profound impact of crystallinity on solid-state fluorescence properties. In particular, we show that stereopure AIEgens form aggregates of superior crystallinity, which in turn exhibit a higher fluorescence quantum yield compared to diastereoisomers mixtures. Preparation of stereopure organic nanoparticles affords very bright fluorescent contrast agents, which are then used for cellular and intravital two-photon microscopy on human breast cancer cells and on zebrafish embryos.

Guanine Radicals Induced in DNA by Low-Energy Photoionization.

Guanine (G) radicals are precursors to DNA oxidative damage, correlated with carcinogenesis and aging. During the past few years, we demonstrated clearly an intriguing effect: G radicals can be generated upon direct absorption of UV radiation with energy significantly lower than the G ionization potential. Using nanosecond transient absorption spectroscopy, we studied the primary species, ejected electrons and guanine radicals, which result from photoionization of various DNA systems in aqueous solution.The DNA propensity to undergo electron detachment at low photon energies greatly depends on its secondary structure. Undetected for monomers or unstacked oligomers, this propensity may be 1 order of magnitude higher for G-quadruplexes than for duplexes. The experimental results suggest nonvertical processes, associated with the relaxation of electronic excited states. Theoretical studies are required to validate the mechanism and determine the factors that come into play. Such a mechanism, which may be operative over a broad excitation wavelength range, explains the occurrence of oxidative damage observed upon UVB and UVA irradiation.Quantification of G radical populations and their time evolution questions some widespread views. It appears that G radicals may be generated with the same probability as pyrimidine dimers, which are considered to be the major lesions induced upon absorption of low-energy UV radiation by DNA. As most radical cations undergo deprotonation, the vast majority of the final reaction products is expected to stem from long-lived deprotonated radicals. Consequently, when G radical cations are involved, the widely used oxidation marker 8-oxodG is not representative of the oxidative damage.Beyond the biological consequences, photogeneration of electron holes in G-quadruplexes may inspire applications in nanoelectronics; although four-stranded structures are currently studied as molecular wires, their behavior as photoconductors has not been explored so far.In the present Account, after highlighting some key experimental issues, we first describe the photoionization process, and then, we focus on radicals. We use as show-cases new results obtained for genomic DNA and Oxytricha G-quadruplexes. Generation and reaction dynamics of G radicals in these systems provide a representative picture of the phenomena reported previously for duplexes and G-quadruplexes, respectively.

Guanine Radicals Generated in Telomeric G-Quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions.

The study deals with the primary species, ejected electrons, and guanine radicals, leading to oxidative damage, that is generated in four-stranded DNA structures (guanine quadruplexes) following photo-ionization by low-energy UV radiation. Performed by nanosecond transient absorption spectroscopy with 266 nm excitation, it focusses on quadruplexes formed by folding of GGG(TTAGGG)3 single strands in the presence of K+ ions, TEL21/K+. The quantum yield for one-photon ionization (9.4 × 10-3) was found to be twice as high as that reported previously for TEL21/Na+. The overall population of guanine radicals decayed faster, their half times being, respectively, 1.4 and 6.7 ms. Deprotonation of radical cations extended over four orders of magnitude of time; the faster step, concerning 40% of their population, was completed within 500 ns. A reaction intermediate, issued from radicals, whose absorption spectrum peaked around 390 nm, was detected.

Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-Quadruplexes and Modify Their Reactivity.

G-Quadruplexes are formed by guanine rich DNA/RNA sequences in the presence of metal ions, which occupy the central cavity of these four-stranded structures. We show that these metal ions have a significant effect on the photogeneration and the reactivity of guanine radicals. Transient absorption experiments on G-quadruplexes formed by association of four TGGGGT strands in the presence of K+ reveal that the quantum yield of one-photon ionization at 266 nm (8.1 × 10-3) is twice as high as that determined in the presence of Na+. Replacement of Na+ with K+ also suppresses one reaction path involving deprotonated radicals, (G-H2)• → (G-H1)• tautomerization. Such behavior shows that the underlying mechanisms are governed by dynamical processes, controlled by the mobility of metal ions, which is higher for Na+ than for K+. These findings may contribute to our understanding of the ultraviolet-induced DNA damage and optimize optoelectronic devices based on four-stranded structures, beyond DNA.

Comprehensive Study of Guanine Excited State Relaxation and Photoreactivity in G-quadruplexes.

G-quadruplexes (G4) are four-stranded DNA/RNA structures playing a key role in many biological functions and promising for nanotechnology applications. Here, combining theoretical calculations and multiscale time-resolved fluorescence, we describe, for the first time, an ensemble of photoactivated processes involving the guanines of the G4 core. We use as showcase the G4 formed by the human telomeric sequence GGG(TTAGGG)3 in the presence of Na+ ions. According to quantum mechanical/molecular mechanics calculations, the hyperchromism at the red part of the absorption spectrum, typical of G4 structures, arises mainly from the inner Na+ ions. Various relaxation pathways, leading to excited states localized on individual bases, neutral excimers, and excited charge transfer states between two guanines or a guanine and a thymine in the loop, are mapped. Their fingerprints are detected in the fluorescence anisotropies and the fluorescence decays, spanning five decades of time. Finally, a reaction funnel leading to guanine dimerization is identified.

Populations and Dynamics of Guanine Radicals in DNA strands-Direct versus Indirect Generation.

Guanine radicals, known to be involved in the damage of the genetic code and aging, are studied by nanosecond transient absorption spectroscopy. They are generated in single, double and four-stranded structures (G-quadruplexes) by one and two-photon ionization at 266 nm, corresponding to a photon energy lower than the ionization potential of nucleobases. The quantum yield of the one-photon process determined for telomeric G-quadruplexes (TEL25/Na+) is (5.2 ± 0.3) × 10-3, significantly higher than that found for duplexes containing in their structure GGG and GG sequences, (2.1 ± 0.4) × 10-3. The radical population is quantified in respect of the ejected electrons. Deprotonation of radical cations gives rise to (G-H1)• and (G-H2)• radicals for duplexes and G-quadruplexes, respectively. The lifetimes of deprotonated radicals determined for a given secondary structure strongly depend on the base sequence. The multiscale non-exponential dynamics of these radicals are discussed in terms of inhomogeneity of the reaction space and continuous conformational motions. The deviation from classical kinetic models developed for homogeneous reaction conditions could also be one reason for discrepancies between the results obtained by photoionization and indirect oxidation, involving a bi-molecular reaction between an oxidant and the nucleic acid.

Radicals Generated in Tetramolecular Guanine Quadruplexes by Photoionization: Spectral and Dynamical Features.

G-quadruplexes are four-stranded DNA structures playing a key role in many biological functions and are promising for applications in the field of nanoelectronics. Characterizing the generation and fate of radical cations (electron holes) within these systems is important in relation to the DNA oxidative damage and/or conductivity issues. This study focuses on guanine radicals in G-quadruplexes formed by association of four TGGGGT strands in the presence of Na+ cations, (TG4T)4/Na+. Using nanosecond transient spectroscopy with 266 nm excitation, we quantitatively characterize hydrated ejected electrons and three types of guanine radicals. We show that, at an energy lower by 2.7 eV than the guanine ionization potential, one-photon ionization occurs with quantum yield of (3.5 ± 0.5) × 10-3. Deprotonation of the radical cations is completed within 20 µs, leading to the formation of (G-H2)• radicals, following a strongly nonexponential decay pattern. Within 10 ms, the latter undergoes tautomerization to deprotonated (G-H1)• radicals. The dynamics of the various radicals determined for (TG4T)4/Na+, in connection to those reported previously for telomeric G-quadruplexes TEL21/Na+, is correlated with energetic factors computed by quantum chemical methods. The faster deprotonation of radical cations in (TG4T)4/Na+ compared to TEL21/Na+ explains that irradiation of the former does not generate 8-oxodGuo, which is readily detected by high-performance liquid chromatography/mass spectrometry in the case of TEL21/Na+.

A "Multi-Heavy-Atom" Approach toward Biphotonic Photosensitizers with Improved Singlet-Oxygen Generation Properties.

Two trispicolinate 1,4,7-triazacyclonane (TACN)-based ligands bearing three picolinate biphotonic antennae were synthetized and their Yb3+ and Gd3+ complexes isolated. One series differs from the other by the absence (L1 )/presence (L2 ) of bromine atoms on the antenna backbone, offering respectively improved optical and singlet-oxygen generation properties. Photophysical properties of the ligands, complexes and micellar Pluronic suspensions were investigated. Complexes exhibit high two-photon absorption cross-section combined either with NIR emission (Yb) or excellent 1 O2 generation (Gd). The very large intersystem crossing efficiency induced by the combination of bromine atom and heavy rare-earth element was corroborated with theoretical calculations. The 1 O2 generation properties of L2 Gd micellar suspension under two-photon activation leads to tumour cell death, suggesting the potential of such structures for theranostic applications.

Radicals generated in alternating guanine-cytosine duplexes by direct absorption of low-energy UV radiation.

Recent studies have evidenced that oxidatively damaged DNA, which potentially leads to carcinogenic mutations and aging, may result from the direct absorption of low-energy photons (>250 nm). Herein, the primary species, i.e., ejected electrons and base radicals associated with such damage in duplexes with an alternating guanine-cytosine sequence are quantified by nanosecond transient absorption spectroscopy. The one-photon ionization quantum yield at 266 nm is 1.2 × 10-3, which is similar to those reported previously for adenine-thymine duplexes. This means that the simple presence of guanine, the nucleobase with the lowest ionization potential, does not affect photo-ionization. The transient species detected after 3 µs are identified as deprotonated guanine radicals, which decay with a half-time of 2.5 ms. Spectral assignment is made with the help of quantum chemistry calculations (TD-DFT), which for the first time, provide reference absorption spectra for guanine radicals in duplexes. In addition, our computed spectra predict the changes in transient absorption expected for hole localization as well as deprotonation (to cytosine and bulk water) and hydration of the radical cation.

Topology Controls the Electronic Absorption and Delocalization of Electron Holes in Guanine Quadruplexes.

Guanine quadruplexes (G4) are four-stranded DNA structures involved in biological processes and are promising candidates for potential nanotechnological applications. This study examines how the G4 topology affects the electronic absorption and the delocalization of electron holes, which play a key role in charge transport and oxidative damage. Combining transient absorption spectroscopy with PCM/TD-DFT calculations both parallel (P) and antiparallel (A) G4 are investigated, which are formed, respectively, by association of four TGGGGT strands and folding of the human telomeric sequence GGG(TTAGGG)3 . The experimental absorption spectra obtained upon photo-ionization of A and P are different. This is explained by the different topology of the two G4, as well as by hole delocalization between two stacked guanines, possible only in P+ . The spectral signature of delocalized hole in guanine-rich regions is provided and the chemical physical effects which rule the hole delocalization are discussed.

Adenine radicals generated in alternating AT duplexes by direct absorption of low-energy UV radiation.

There is increasing evidence that the direct absorption of photons with energies that are lower than the ionization potential of nucleobases may result in oxidative damage to DNA. The present work, which combines nanosecond transient absorption spectroscopy and quantum mechanical calculations, studies this process in alternating adenine-thymine duplexes (AT)n. We show that the one-photon ionization quantum yield of (AT)10 at 266 nm (4.66 eV) is (1.5 ± 0.3) × 10-3. According to our PCM/TD-DFT calculations carried out on model duplexes composed of two base pairs, (AT)1 and (TA)1, simultaneous base pairing and stacking does not induce important changes in the absorption spectra of the adenine radical cation and deprotonated radical. The adenine radicals, thus identified in the time-resolved spectra, disappear with a lifetime of 2.5 ms, giving rise to a reaction product that absorbs at 350 nm. In parallel, the fingerprint of reaction intermediates other than radicals, formed directly from singlet excited states and assigned to AT/TA dimers, is detected at shorter wavelengths. PCM/TD-DFT calculations are carried out to map the pathways leading to such species and to characterize their absorption spectra; we find that, in addition to the path leading to the well-known TA* photoproduct, an AT photo-dimerization path may be operative in duplexes.

UV-induced damage to DNA: effect of cytosine methylation on pyrimidine dimerization.

Methylation/demethylation of cytosine plays an important role in epigenetic signaling, the reversibility of epigenetic modifications offering important opportunities for targeted therapies. Actually, methylated sites have been correlated with mutational hotspots detected in skin cancers. The present brief review discusses the physicochemical parameters underlying the specific ultraviolet-induced reactivity of methylated cytosine. It focuses on dimerization reactions giving rise to cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone adducts. According to recent studies, four conformational and electronic factors that are affected by cytosine methylation may control these reactions: the red-shift of the absorption spectrum, the lengthening of the excited state lifetime, changes in the sugar puckering modifying the stacking between reactive pyrimidines and an increase in the rigidity of duplexes favoring excitation energy transfer toward methylated pyrimidines.

Absorption of Low-Energy UV Radiation by Human Telomere G-Quadruplexes Generates Long-Lived Guanine Radical Cations.

Telomeres, which are involved in cell division, carcinogenesis, and aging and constitute important therapeutic targets, are prone to oxidative damage. This propensity has been correlated with the presence of guanine-rich sequences, capable of forming four-stranded DNA structures (G-quadruplexes). Here, we present the first study on oxidative damage of human telomere G-quadruplexes without mediation of external molecules. Our investigation has been performed for G-quadruplexes formed by folding of GGG(TTAGGG)3 single strands in buffered solutions containing Na+ cations (TEL21/Na+). Associating nanosecond time-resolved spectroscopy and quantum mechanical calculations (TD-DFT), it focuses on the primary species, ejected electrons and guanine radicals, generated upon absorption of UV radiation directly by TEL21/Na+. We show that, at 266 nm, corresponding to an energy significantly lower than the guanine ionization potential, the one-photon ionization quantum yield is 4.5 × 10-3. This value is comparable to that of cyclobutane thymine dimers (the major UV-induced lesions) in genomic DNA; the quantum yield of these dimers in TEL21/Na+ is found to be (1.1 ± 0.1) × 10-3. The fate of guanine radicals, generated in equivalent concentration with that of ejected electrons, is followed over 5 orders of magnitude of time. Such a quantitative approach reveals that an important part of radical cation population survives up to a few milliseconds, whereas radical cations produced by chemical oxidants in various DNA systems are known to deprotonate, at most, within a few microseconds. Under the same experimental conditions, neither one-photon ionization nor long-lived radical cations are detected for the telomere repeat TTAGGG in single-stranded configuration, showing that secondary structure plays a key role in these processes. Finally, two types of deprotonated radicals are identified: on the one hand, (G-H2)• radicals, stable at early times, and on the other hand, (G-H1)• radicals, appearing within a few milliseconds and decaying with a time constant of ∼50 ms.