Marco Agnes

1-Hydroxypyrene (1HOPy) is the most widely used urinary biomarker for monitoring human exposure to polycyclic aromatic hydrocarbons (PAHs). Current analytical methods, primarily based on chromatographic separation, require sophisticated instrumentation and sample pre-treatment, limiting in situ analysis. Here we investigate cyclodextrin (CD)-based supramolecular platforms as molecular recognition scaffolds for fluorescence-based detection of 1HOPy. Six CD derivatives, including two monomers, hydroxypropyl-βCD (HPβCD), and γCD, two pβCD and pγCD polymers (pβCD), and two synthetic βCD dimers (βDim1, βDim2), were systematically evaluated through UV-Vis absorption, steady-state and time-resolved fluorescence, and molecular dynamics simulations coupled with thermodynamic integration free energy calculations. Host-dependent complexation stoichiometries (1:1, 2:1, and 2:2) and binding constants spanning from 103 to 105M⁻¹ for 1:1 complexes were obtained via multivariate global analysis. HPβCD exhibited the highest 1:1 binding affinity (K1:1 = 1.0×105 M-1) with moderate fluorescence enhancement. Differently, γCD formed cooperative 2:2 assemblies displaying pyrene excimer emission at 480 nm. Most notably, the primary-side βCD dimer βDim2 promoted ground-state deprotonation of 1HOPy upon complexation (K1:1 = 1.2×104 M-1), generating a phenolate species with a strongly blue-shifted fluorescence that is spectroscopically distinct from both free 1HOPy and other common PAH metabolites. Computational analysis, combining 100-ns molecular dynamics trajectories with thermodynamic integration binding free energies, fairly reproduced the experimental affinity hierarchy. Preliminary tests demonstrated that 1HOPy excitation at 400 nm in the presence of 1 mM βDim2 enables exclusive detection of 1HOPy without interference from co-occurring PAH metabolites (1-naphthol, 2-naphthol, 2-fluorenol, 9-fluorenol), with a linear fluorescence response over the 10-1000 nM range. These findings establish βDim2 as a promising molecular recognition element for sensitive, selective, and potentially field-deployable fluorimetric detection of 1HOPy as a PAH biomarker.

Hypoxia occurs in different pathological settings as a consequence of poor vascularization, and it results in reduced efficacy of some current nosocomial treatments i. e. chemotherapy and photodynamic therapy. In order to overcome these boundaries, aromatic endoperoxides (EPOs) have been studied and proposed as oxygen‐releasing agents (ORAs) due to their ability to reversibly bind molecular oxygen (O₂), liberating it upon suitable triggering. DFT calculation of the dissociation energy (E_diss) of the intramolecular O−O bridge and structural crystallographic data of synthesized and studied EPOs drove the design of an array of 9,10‐disubstituted anthracenes among which three candidates were carefully selected. Once optimized the synthesis of the aromatic substrates, for the first time the corresponding EPOs have been produced under continuous flow irradiation in the presence of a sub‐stoichiometric amount of a photosensitizer in organic solvents. The release of O₂ could be obtained straightforwardly at 37.5 °C by thermolysis. In accordance with the calculated E_diss=3.2 kcal mol⁻¹ and an experimental t_1/2=40 minutes, 3,3′‐(anthracene‐9,10‐diyl)bis(prop‐2‐yn‐1‐ol) resulted as the best candidate for the sustained release of O₂ under physiologically relevant conditions. Its exploitation as ORA to relieve hypoxia will be evaluated and described in due course.

Hypoxia occurs in different pathological settings as a consequence of poor vascularization and it results in reduced efficacy of current nosocomial treatments i.e. chemotherapy and photodynamic therapy. In order to overcome these boundaries, aromatic endoperoxides (EPOs) have been studied and proposed as oxygen-releasing agents (ORAs) due to their ability to reversibly bind molecular oxygen (O2), liberating it upon suitable triggering. DFT calculation of the dissociation energy (Ediss) of the intramolecular O-O bridge and structural crystallographic data of synthesized and studied EPOs drove the design of an array of 9,10-disubstituted anthracenes among which three candidates were carefully selected. Once optimized the synthesis of the aromatic substrates, for the first time the corresponding EPOs have been produced under continuous flow irradiation in the presence of a sub-stoichiometric amount of a photosensitizer in organic solvents. The release of O2 could be obtained straightforwardly at 37.5 °C by thermolysis. In accordance with the calculated Ediss = 3.2 kcal mol-1, 3,3'-(anthracene-9,10-diyl)bis(prop-2-yn-1-ol) (DIPA) resulted as the best candidate for the sustained release of O2 in biocompatible and physiologically relevant conditions. The exploitation of these optimized EPOs as ORAs to relieve hypoxia will be further evaluated and described in due course.

Versatile water-soluble cyclodextrin polymers can be easily prepared from naturally occurring monomers upon polycondensation in the presence of crosslinking agents. We consider their potential use in combination therapies and the hurdles faced.

We explored a series of cyclodextrin (CyD) polymers composed either of a single CyD type or a mixture of two CyD types to encapsulate simultaneously different compounds with potential therapeutic interest for multimodal prostate cancer treatment. New mixed CyD polymers were prepared in alkaline water starting from the naturally occurring monomers and a low-cost crosslinking agent. Batches of 200 g of polymer were easily obtained. By means of optical spectroscopy we proved the co-encapsulation of 3 compounds in the polymers: the drugs cabazitaxel (CBX) and bicalutamide (BIC), and the photosensitizer chlorin e6 (Ce6). pβCyD and mixed pαβCyD polymers performed best for single drug solubilization. In the co-encapsulation of BIC and CBX by pβCyD and pαβCyD, pβCyD stands out in drug solubilization ability. Avoiding the use of organic solvents, it was possible to dissolve up to 0.1 mM CBX with 10 mg ml⁻¹ pβCyD polymer and, with 100 mg ml⁻¹, even 1.7 mM BIC, a 100-fold improvement compared to water. Spectroscopic studies afforded the binding constants of CBX and BIC with pβCyD forming complexes of 1 : 2 stoichiometry (drug : CyD) and CBX displayed significantly higher affinity. Also DFT calculations suggested that the drugs are more stable when complexed by two CyD units. Ce6 could be encapsulated simultaneously with the other two drugs in pβCyD and, most importantly, is able to produce singlet oxygen efficiently. Thanks to a single inexpensive CyD-based polymer we were able to produce a three-in-one platform for future implementation of combined chemotherapy and photodynamic therapy. These achievements are most relevant as nanomedicines are continuously proposed but their potential for translation to the pharma industry is compromised by their limited potential for industrial upscale.

In the quest for new therapies targeting hypoxia, aromatic endoperoxides have intriguing potential as oxygen releasing agents (ORAs) able to free O₂ in tissues upon suitable trigger. Four aromatic substrates were synthesized and the formation of their corresponding endoperoxides was optimized in organic solvent upon selective irradiation of Methylene Blue, a low‐cost photocatalyst, producing the reactive singlet oxygen species. Complexation of the hydrophobic substrates within a hydrophilic cyclodextrin (CyD) polymer allowed their photooxygenation in homogeneous aqueous environment using the same optimized protocol upon dissolution in water of the three readily accessible reagents. Notably, reaction rates were comparable in buffered D₂O and organic solvent and, for the first time, the photooxygenation of highly hydrophobic substrates was achieved for millimolar solutions in non‐deuterated water. Quantitative conversion of the substrates, straightforward isolation of the endoperoxides and recovery of the polymeric matrix were achieved. Cycloreversion of one ORA to the original aromatic substrate was observed upon thermolysis. These results hold great potential for the launch of CyD polymers both as reaction vessels for green, homogeneous photocatalysis and as carrier for the delivery of ORAs in tissues.

Cyclodextrin‐based polymers can be prepared starting from the naturally occurring monomers following green and low‐cost procedures. They can be selectively derivatized pre‐ or post‐polymerization allowing to fine‐tune functionalities of ad hoc customized polymers. Preparation nowadays has reached the 100 g scale thanks also to the interest of industries in these extremely versatile compounds. During the last 15 years, these macromolecules have been the object of intense investigations in view of possible biomedical applications as the ultimate goal and large amounts of scientific data are now available. Compared to their monomeric models, already used in the formulation of various therapeutic agents, they display superior behavior in terms of their solubility in water and solubilizing power toward drugs incompatible with biological fluids. Moreover, they allow the combination of more than one type of therapeutic agent in the polymeric system. In this review, a complete state of the art on the knowledge and potentialities of water‐soluble cyclodextrin‐based polymers as therapeutic agents as well as carrier systems for different types of therapeutics to implement combination therapy is provided. Finally, a perspective on their assets for innovation in disease treatment as well as their limits that still need to be addressed is given.