Edurne Nuin

Photophysical Probes To Assess the Potential of Cholic Acid Aggregates as Drug Carriers

The two enantiomers of the nonsteroidal antiinflammatory drug naproxen and of its methyl ester have been selected as representative probes with markedly different hydrophobicity to assess the potential of cholic acid aggregates as drug carriers by means of photophysical techniques. The different distribution of the probes between bulk solution and aggregates has been assessed by quenching of their singlet and triplet excited states by iodide and nitrite anions, respectively. This straightforward photophysical methodology can, in principle, be extended to a variety of drugs containing a photoactive chromophore.

Fluoroquinolone Photodegradation Influences Specific Basophil Activation

Fluoroquinolones (FQs) are photoreactive drugs, but it is not known whether laboratory light exposure can influence the induction of photoproducts and modify in vitro test results. The basophil activation test (BAT) has proven to be useful for evaluating immunoglobulin E (IgE)-mediated hypersensitivity to FQs, with a higher percentage of positive responders with ciprofloxacin (CIP) than with moxifloxacin (MOX). We studied the effect of laboratory light on CIP and MOX degradation, and drug-protein conjugate formation, and its influence on the BAT for evaluating IgE-mediated hypersensitivity to FQs. The results showed an important decrease in fluorescence emission intensity under light compared to dark conditions for MOX, and that BAT positivity was lower in light (17.9%) than in dark (35.7%) conditions. No changes were found for CIP in either fluorescence emission intensity or BAT results (46.4% in both conditions). We can conclude that light exposure is a critical factor in BAT results when photolabile drugs like MOX are used. Therefore, light is important when interpreting in vitro results.

Enhanced photosafety of cinacalcet upon complexation with serum albumin.

Cinacalcet (CIN) is a calcimimetic drug, which contains a naphthalene chromophore and binds almost quantitatively to human serum albumin (HSA). In the present work, the excited states of CIN have been characterized in order to obtain relevant information about complexation of CIN with HSA. The fluorescence spectrum in acetonitrile, at λ(exc) = 290 nm, displayed two bands with maxima at 332 and 439 nm, assigned to the monomer and exciplex emission. Upon protonation of the amino group, the exciplex band disappeared, with a concomitant increase of the monomer emission intensity. Time-resolved fluorescence evidenced an intramolecular dynamic quenching, attributed to exciplex formation and/or photoinduced electron transfer, in agreement with the favorable thermodynamics predicted by the Rehm-Weller equations. Diffusion controlled dynamic quenching of CINH(+) fluorescence by oxygen was observed. The emission properties in PBS were similar to those obtained for CINH(+) in acetonitrile. Laser flash photolysis (LFP) of CIN and CINH(+) in acetonitrile/N(2), at λ(exc) = 308 nm, gave rise to the naphthalene-like triplet excited states, with maxima at 420 nm and lifetimes of 4 and 7 μs; they were efficiently quenched by oxygen. No significant singlet excited state interaction was observed in CINH(+)/HSA complexes, as revealed by the emission spectra, which were roughly explained taking into account the relative contributions of drug and protein in the absorption spectra. Upon LFP of the complexes, triplet excited states were generated; the decays monitored at 420 nm were satisfactorily fitted using a function containing two monoexponential terms, corresponding to a short-lived (τ(1) = 8 μs) and a long-lived (τ(2) = 37 μs) component. This indicates that the drug is incorporated into two different binding sites of HSA. Despite the long triplet lifetimes of the CINH(+)/HSA complexes, the rate constant of quenching by oxygen was found to be 2 orders of magnitude lower than that determined in acetonitrile, which can be attributed to the relative slower diffusion rates in this microheterogeneous system. Therefore, the protein microenvironment protects cinacalcet from attack by oxygen; this prevents the phototoxic effects caused by formation of singlet oxygen and results in an enhanced photosafety of the drug.

Naphthalene triplet excited state as a probe for the assessment of drug distribution in binary protein systems.

Three drugs containing the naphthalene (NP) chromophore, namely, naproxen (NPX), propranolol (PPN), and cinacalcet (CIN), but with different affinities toward serum albumins (SAs) and α-1-acid glycoproteins (AAGs) have been employed for the assessment of drug distribution in binary SA/AAG systems. These three drugs represent an appropriate choice for checking whether a methodology based on transient absorption spectroscopy of a given reporter can be employed for discrimination between different distribution patterns in multicompartmental biological media. Thus, upon laser flash photolysis (LFP) of NPX, PPN, and CIN in the presence or absence of proteins, the NP triplet excited state ((3)NP*) at ∼420 nm was always detected, although the kinetics of the decay traces was structure- and medium-dependent. In aerated PBS, only a very short triplet lifetime (τ(T)) was found (1-2 μs). By contrast, in the presence of SAs, two longer triplet lifetimes (5-76 μs) were observed, ascribed to (3)NP* within site I and site II. Upon binding to AAGs, only a long τ(T) (15-47 μs) was found. When the two proteins were present simultaneously in the same media, fitting of the decay traces was clearly consistent with a distribution of the drug between the different biological compartments and the bulk solution, which correlates well with the known protein affinities of every drug. Experiments were performed in both human (HSA/HAAG) and bovine protein media (BSA/BAAG). The results showed that SAs are the major carriers for NPX; by contrast, PPN binds preferentially to AAGs. An intermediate situation was found for CIN, which presents comparable affinity for both proteins. The results obtained for the two enantiomers of each drug were very similar, although a small stereodifferentiation was observed between the triplet lifetimes in the protein binding sites.

New photoactive compounds to probe cholic acid and cholesterol inside mixed micelles

New photoactive dyads have been synthesized by derivatization of cholic acid (CA) or cholesterol (Ch). These compounds have proven to be efficient tools to monitor incorporation of CA and Ch to mixed micelles (MM) and to probe the microenvironment experienced inside these entities. The outstanding capability of MM to solubilize Ch has been demonstrated.

Noncovalent Functionalization and Charge Transfer in Antimonene

Abstract Antimonene, a novel group 15 two‐dimensional material, is functionalized with a tailormade perylene bisimide through strong van der Waals interactions. The functionalization process leads to a significant quenching of the perylene fluorescence, and surpasses that observed for either graphene or black phosphorus, thus allowing straightforward characterization of the flakes by scanning Raman microscopy. Furthermore, scanning photoelectron microscopy studies and theoretical calculations reveal a remarkable charge‐transfer behavior, being twice that of black phosphorus. Moreover, the excellent stability under environmental conditions of pristine antimonene has been tackled, thus pointing towards the spontaneous formation of a sub‐nanometric oxide passivation layer. DFT calculations revealed that the noncovalent functionalization of antimonene results in a charge‐transfer band gap of 1.1 eV.

Influence of drug encapsulation within mixed micelles on the excited state dynamics and accessibility to ionic quenchers.

Photophysical techniques, specifically time-resolved fluorescence and laser flash photolysis, have proven to be noninvasive, straightforward, and valuable tools to demonstrate how drug encapsulation into biomimetic mixed micelles (MM) influences the dynamics of excited states and their accessibility to ionic quenchers. This concept has been illustrated by choosing a set of currently administered drugs containing a common naphthalene chromophore, namely, (S)-naproxen and its methyl ester, (R)-cinacalcet and (S)-propranolol. A remarkable increase of their triplet lifetimes is noticed when experiments are performed in MM, indicating efficient entrapment of the drugs in these supramolecular entities. Furthermore, a decrease of 1 order of magnitude in the quenching rate constant of the singlet and triplet excited states (by iodide or nitrite, respectively) is observed upon encapsulation into MM. This approach can in principle be extended to other microenvironments capable of incorporating photoactive compounds.

Drug-drug interactions within protein cavities probed by triplet-triplet energy transfer

A new direct and noninvasive methodology based on transient absorption spectroscopy has been developed to probe the feasibility of drug-drug interactions within a common protein binding site. The simultaneous presence of (R)-cinacalcet (CIN) and (S)-propranolol (PPN) within human or bovine α1-acid glycoproteins (AAGs) is revealed by detection of (3)CIN* as the only transient species after laser flash photolysis of CIN/PPN/AAG mixtures at 308 nm. This is the result of triplet-triplet energy transfer from (3)PPN* to CIN, which requires close contact between the two drugs within the same biological compartment. Similar results are obtained with nabumetone and CIN as donor/acceptor partners. This new methodology can, in principle, be extended to a variety of drug/drug/biomolecule combinations.

Time-Resolved Fluorescence Study of Exciplex Formation in Diastereomeric Naproxen−Pyrrolidine Dyads

The influence of chirality on the elementary processes triggered by excitation of the (S,S)- and (R,S)- diastereoisomers of naproxen-pyrrolidine (NPX-Pyr) dyads has been studied by time-resolved fluorescence in acetonitrile-benzene mixtures. In these systems, the quenching of the (1)NPX*-Pyr singlet excited state occurs through electron transfer and exciplex formation. Fluorescence lifetimes and quantum yields revealed a significant difference (around 20%) between the (S,S)- and (R,S)- diastereomers. In addition, the quantum yields of exciplexes differed by a factor of 2 regardless of solvent polarity. This allows us to suggest a similar influence of the chiral centers on the local charge transfer resulting in exciplex and full charge separation that leads to ion-biradicals. A simplified scheme is proposed to estimate a set of rate constant values (k1-k5) for the elementary stages in each solvent system.

Photosensitivity to Triflusal: Formation of a Photoadduct with Ubiquitin Demonstrated by Photophysical and Proteomic Techniques

Triflusal is a platelet aggregation inhibitor chemically related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacological point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clinical profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint analysis. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction. With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradiation, combining proteomic and photophysical (fluorescence and laser flash photolysis) techniques. Photophysical and proteomic analysis demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addition can take place at any of the ε-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle occur with other trifluoroaromatic compounds and may be responsible for the appearance of undesired photoallergic side effects.