Supramolecular red-light-photosensitized nitric oxide release with fluorescence self-reporting within biocompatible nanocarriers.

The strict dependence of the biological effects of nitric oxide (NO) on its concentration and generation site requires this inorganic free radical to be delivered with precise spatiotemporal control. Light-activation by suitable NO photoprecursors represents an ideal approach. Developing strategies to activate NO release using long-wavelength excitation light in the therapeutic window (650-1300 nm) is challenging. In this contribution, we demonstrate that NO release by a blue-light activatable NO photodonor (NOPD) with self-fluorescence reporting can be triggered catalytically by the much more biocompatible red light exploiting a supramolecular photosensitization process. Different red-light absorbing photosensitizers (PSs) are co-entrapped with the NOPD within different biocompatible nanocarriers such as Pluronic® micelles, microemulsions and branched cyclodextrin polymers. The intra-carrier photosensitized NO release, involving the lowest, long-lived triplet state of the PS as the key intermediate and its quenching by the NOPD, is competitive with that by molecular oxygen. This allows NO to be released with good efficacy, even under aerobic conditions. Therefore, the adopted general strategy provides a valuable tool for generating NO from an already available NOPD, otherwise activatable with the poorly biocompatible blue light, without requiring any chemical modification and using sophisticated and expensive irradiation sources.

Toward Nano- and Microplastic Sensors: Identification of Nano- and Microplastic Particles via Artificial Intelligence Combined with a Plasmonic Probe Functionalized with an Estrogen Receptor.

Nano- and microplastic particles are a global and emerging environmental issue that might pose potential threats to human health. The present work exploits artificial intelligence (AI) to identify nano- and microplastics in water by monitoring the interaction of the sample with a sensitive surface. An estrogen receptor (ER) grafted onto a gold surface, realized on a nonexpensive and easy-to-produce plastic optical fiber (POF) platform in order to excite a surface plasmon resonance (SPR) phenomenon, has been developed in order to carry out a "smart" sensitive interface (ER-SPR-POF interface). The ER-SPR-POF interface offers output data useful for exploiting a machine learning-based approach to achieve nano- and microplastic particle sensors. This work developed a proof-of-concept sensor through a training phase carried out by different particles, in terms of materials and size. The experimental results have demonstrated that the proposed "smart" ER-SPR-POF interface combined with AI can be used to identify the kind of particles in terms of the materials (polystyrene; poly(methyl methacrylate)) and size (20 µm; 100 nm) with an accuracy of 90.3%.

Mixed ß-γ-Cyclodextrin Branched Polymer with Multiple Photo-Chemotherapeutic Cargos.

The achievement of biocompatible platforms for multimodal therapies is one of the major challenges in the burgeoning field of nanomedicine. Here, we report on a mixed ß- and γ-cyclodextrin-based branched polymeric material (ßγCD-NOPD) covalently integrating a nitric oxide (NO) photodonor (NOPD) within its macromolecular scaffold, and its supramolecular ensemble with a singlet oxygen (1O2) photosensitizer (PS) Zn(II) phthalocyanine (ZnPc) and the chemodrug Lenvatinib (LVB). This polymer is highly water-soluble and generates NO under visible blue light stimuli with an efficiency of more than 1 order of magnitude higher than that of the single NOPD. The PS, which in an aqueous solution is aggregated and non-photoresponsive, can be entangled in the polymeric network as a photoresponsive monomeric species. In addition, the poorly water-soluble LVB can be co-encapsulated within the polymeric host, which increases the drug solubility by more than 30-fold compared to the free drug and more than 2-fold compared with a similar branched polymer containing only ßCD units. The supramolecular nanoensemble, ca. 15 nm in diameter, retains well the photochemical properties of both the NOPD and PS, which can operate in parallel under light stimuli of different energies. Irradiation with blue and red light results in the photogeneration of NO and 1O2 associated with red fluorescence emission, without inducing any photodegradation of LVB. This result is not trivial and is due to the absence of significant, mutual interactions between the NOPD, the PS and LVB both in the ground and excited states, despite these components are confined in the same host. The proposed polymeric nanoplatform may represent a potential trimodal nanomedicine for biomedical research studies, since it combines the double photodynamic action of NO and 1O2, two species that do not suffer multidrug resistance, with the therapeutic activity of a conventional chemodrug.

Sensing Approaches Exploiting Molecularly Imprinted Nanoparticles and Lossy Mode Resonance in Polymer Optical Fibers.

In this work, two different lossy mode resonance (LMR) platforms based on plastic optical fibers (POFs) are developed and tested in a biochemical sensing scenario. The LMR platforms are based on the combination of two metal oxides (MOs), i.e., zirconium oxide (ZrO2) and titanium oxide (TiO2), and deposited on the exposed core of D-shaped POF chips. More specifically, two experimental sensor configurations were obtained by swapping the mutual position of the Mos films over to the core of the D-shaped POF probe. The POF-LMR sensors were first characterized as refractometers, proving the bulk sensitivities. Then, both the POF-LMR platforms were functionalized using molecularly imprinted nanoparticles (nanoMIPs) specific for human transferrin (HTR) in order to carry out binding tests. The achieved results report a bulk sensitivity equal to about 148 nm/RIU in the best sensor configuration, namely the POF-TiO2-ZrO2. In contrast, both optical configurations combined with nanoMIPs showed an ultra-low detection limit (fM), demonstrating excellent efficiency of the used receptor (nanoMIPs) and paving the way to disposable POF-LMR biochemical sensors that are easy-to-use, low-cost, and highly sensitive.

Soft molecularly imprinted nanoparticles with simultaneous lossy mode and surface plasmon multi-resonances for femtomolar sensing of serum transferrin protein.

The simultaneous interrogation of both lossy mode (LMR) and surface plasmon (SPR) resonances was herein exploited for the first time to devise a sensor in combination with soft molecularly imprinting of nanoparticles (nanoMIPs), specifically entailed of the selectivity towards the protein biomarker human serum transferrin (HTR). Two distinct metal-oxide bilayers, i.e. TiO2-ZrO2 and ZrO2-TiO2, were used in the SPR-LMR sensing platforms. The responses to binding of the target protein HTR of both sensing configurations (TiO2-ZrO2-Au-nanoMIPs, ZrO2-TiO2-Au-nanoMIPs) showed femtomolar HTR detection, LODs of tens of fM and KDapp ~ 30 fM. Selectivity for HTR was demonstrated. The SPR interrogation was more efficient for the ZrO2-TiO2-Au-nanoMIPs configuration (sensitivity at low concentrations, S = 0.108 nm/fM) than for the TiO2-ZrO2-Au-nanoMIPs one (S = 0.061 nm/fM); while LMR was more efficient for TiO2-ZrO2-Au-nanoMIPs (S = 0.396 nm/fM) than for ZrO2-TiO2-Au-nanoMIPs (S = 0.177 nm/fM). The simultaneous resonance monitoring is advantageous for point of care determinations, both in terms of measurement's redundancy, that enables the cross-control of the measure and the optimization of the detection, by exploiting the individual characteristics of each resonance.

Estradiol Detection for Aquaculture Exploiting Plasmonic Spoon-Shaped Biosensors.

In this work, a surface plasmon resonance (SPR) biosensor based on a spoon-shaped waveguide combined with an estrogen receptor (ERα) was developed and characterized for the detection and the quantification of estradiol in real water samples. The fabrication process for realizing the SPR platform required a single step consisting of metal deposition on the surface of a polystyrene spoon-shaped waveguide featuring a built-in measuring cell. The biosensor was achieved by functionalizing the bowl sensitive surface with a specific estrogen receptor (ERα) that was able to bind the estradiol. In a first phase, the biosensor tests were performed in a phosphate buffer solution obtaining a limit of detection (LOD) equal to 0.1 pM. Then, in order to evaluate the biosensor's response in different real matrices related to aquaculture, its performances were examined in seawater and freshwater. The experimental results support the possibility of using the ERα-based biosensor for the screening of estradiol in both matrices.

Green Synthesis of Near-Infrared Plasmonic Gold Nanostructures by Pomegranate Extract and Their Supramolecular Assembling with Chemo- and Photo-Therapeutics.

Au nanostructures exhibiting a localized surface plasmon resonance in the near-infrared spectral window are obtained in a single, green step at room temperature by pomegranate extract in the presence of a highly biocompatible ß-cyclodextrin branched polymer, without the need of preformed seeds, external reducing and sacrificial agents, and conventional surfactants. The polymeric component makes the Au nanostructures dispersible in water, stable for weeks and permits their supramolecular assembling with the chemotherapeutic sorafenib and a nitric oxide (NO) photodonor (NOPD), chosen as representative for chemo- and photo-therapeutics. Irradiation of the plasmonic Au nanostructures in the therapeutic window with 808 nm laser light results in a good photothermal response, which (i) is not affected by the presence of either the chemo- or the phototherapeutic guests and (ii) does not lead to their photoinduced decomposition. Besides, irradiation of the hybrid Au nanoassembly with the highly biocompatible green light results in the NO release from the NOPD with efficiency similar to that observed for the free guest. Preliminary biological experiments against Hep-G2 hepatocarcinoma cell lines are also reported.

Spoon-shaped polymer waveguides to excite multiple plasmonic phenomena: A multisensor based on antibody and molecularly imprinted nanoparticles to detect albumin concentrations over eight orders of magnitude.

A polymeric multimode waveguide, characterized by a pioneering spoon-shaped geometry, was herein proposed for the first time to devise Surface Plasmon Resonance (SPR) biochemical sensors. The plasmon excitation was enabled by layering a gold nanofilm of ∼60 nm onto the spoon-waveguide. As a consequence of the waveguide's extra-ordinary geometry, two distinct sensing regions were identified: a planar one, located on the spoon's neck, and a concave one on the bowl, with angled surfaces. The bulk sensitivity (Sn) is correlated both to the way the light was launched in/collected from the sensor (parallel or orthogonal to the main axis of the waveguide) and to the sensing area interrogated (planar-neck or angled-bowl), indicating that the sensor's performance can be conveniently tuned, depending on the chosen measuring configuration. The SPR sensor's characterization showed Sn equal to 750 nm/RIU for the neck and to 950 nm/RIU for the bowl. To further inspect the peculiar sensing-features and assess the application niches, the spoon-shaped waveguide was functionalized with two kinds of receptors, both specific for human serum albumin (HSA): an antibody on the bowl region (high Sn); molecularly imprinted nanoparticles (nanoMIPs) on the neck region (low Sn). The experimental results showed a limit of detection (LOD) for the immune-sensor of 280 pM and an LOD for the nanoMIP-sensor of 4.16 fM. The overall response of the HSA multi-sensor encompassed eight orders of magnitude, suggesting that the spoon-shaped waveguide's provides multi-scale detection and holds potential to devise multi-analyte sensing platforms.

A Plasmonic Biosensor Based on Light-Diffusing Fibers Functionalized with Molecularly Imprinted Nanoparticles for Ultralow Sensing of Proteins.

Plasmonic bio/chemical sensing based on optical fibers combined with molecularly imprinted nanoparticles (nanoMIPs), which are polymeric receptors prepared by a template-assisted synthesis, has been demonstrated as a powerful method to attain ultra-low detection limits, particularly when exploiting soft nanoMIPs, which are known to deform upon analyte binding. This work presents the development of a surface plasmon resonance (SPR) sensor in silica light-diffusing fibers (LDFs) functionalized with a specific nanoMIP receptor, entailed for the recognition of the protein human serum transferrin (HTR). Despite their great versatility, to date only SPR-LFDs functionalized with antibodies have been reported. Here, the innovative combination of an SPR-LFD platform and nanoMIPs led to the development of a sensor with an ultra-low limit of detection (LOD), equal to about 4 fM, and selective for its target analyte HTR. It is worth noting that the SPR-LDF-nanoMIP sensor was mounted within a specially designed 3D-printed holder yielding a measurement cell suitable for a rapid and reliable setup, and easy for the scaling up of the measurements. Moreover, the fabrication process to realize the SPR platform is minimal, requiring only a metal deposition step.

Enhancing the Anticancer Activity of Sorafenib through Its Combination with a Nitric Oxide Photodelivering ß-Cyclodextrin Polymer.

In this contribution, we report a strategy to enhance the therapeutic action of the chemotherapeutic Sorafenib (SRB) through its combination with a multifunctional ß-cyclodextrin-based polymer able to deliver nitric oxide (NO) and emit green fluorescence upon visible light excitation (PolyCDNO). The basically water-insoluble SRB is effectively encapsulated in the polymeric host (1 mg mL-1) up to a concentration of 18 µg mL-1. The resulting host-guest supramolecular complex is able to release SRB in sink conditions and to preserve very well the photophysical and photochemical properties of the free PolyCDNO, as demonstrated by the similar values of the NO release and fluorescence emission quantum efficiencies found. The complex PolyCDNO/SRB internalizes in HEP-G2 hepatocarcinoma, MCF-7 breast cancer and ACHN kidney adenocarcinoma cells, localizing in all cases mainly at the cytoplasmic level. Biological experiments have been performed at SRB concentrations below the IC50 and with light doses producing NO at nontoxic concentrations. The results demonstrate exceptional mortality levels for PolyCDNO/SRB upon visible light irradiation in all the different cell lines tested, indicating a clear synergistic action between the chemotherapeutic drug and the NO. These findings can open up exciting avenues to potentiate the anticancer action of SRB and, in principle, to reduce its side effects through its use at low dosages when in combination with the photo-regulated release of NO.

Single Isomer N-Heterocyclic Cyclodextrin Derivatives as Chiral Selectors in Capillary Electrophoresis.

In order to better understand the chiral recognition mechanisms of positively charged cyclodextrin (CD) derivatives, the synthesis, the pKa determination by 1H nuclear magnetic resonance (NMR)-pH titration and a comparative chiral capillary electrophoretic (CE) study were performed with two series of mono-substituted cationic single isomer CDs. The first series of selectors were mono-(6-N-pyrrolidine-6-deoxy)-ß-CD (PYR-ß-CD), mono-(6-N-piperidine-6-deoxy)-ß-CD (PIP-ß-CD), mono-(6-N-morpholine-6-deoxy)-ß-CD (MO-ß-CD) and mono-(6-N-piperazine-6-deoxy)-ß-CD (PIPA-ß-CD), carrying a pH-adjustable moiety at the narrower rim of the cavity, while the second set represented by their quaternarized, permanently cationic counterparts: mono-(6-N-(N-methyl-pyrrolidine)-6-deoxy)-ß-CD (MePYR-ß-CD), mono-(6-N-(N-methyl-piperidine)-6-deoxy)-ß-CD (MePIP-ß-CD), mono-(6-N-(N-methyl-morpholine)-6-deoxy)-ß-CD (MeMO-ß-CD) and mono-(6-N-(4,4-N,N-dimethyl-piperazine)-ß-CD (diMePIPA-ß-CD). Based on pH-dependent and selector concentration-dependent comparative studies of these single isomer N-heterocyclic CDs presented herein, it can be concluded that all CDs could successfully be applied as chiral selectors for the enantiodiscrimination of several negatively charged and zwitterionic model racemates. The substituent-dependent enantiomer migration order reversal of dansylated-valine using PIP-ß-CD contrary to PYP-ß-CD, MO-ß-CD and PIPA-ß-CD was also studied by 1H- and 2D ROESY NMR experiments.

Nitric Oxide Photoreleasers with Fluorescent Reporting.

Nitric oxide (NO) plays a multifaceted role in human physiology and pathophysiology, and its controlled delivery has great prospects in therapeutic applications. The light-activated uncaging of NO from NO caging compounds allows this free radical to be released with accurate control of site and dosage, which strictly determine its biological effects. Molecular constructs able to activate fluorescence concomitantly to NO release offer the important advantage of easy and real-time tracking of the amount of NO uncaged in a non-invasive fashion even in the cell environment. This contribution provides an overview of the advances in photoactivatable NO releasers bearing fluorescent reporting functionalities achieved in our and other laboratories, highlighting the rationale design and their potential therapeutic applications.

A thermoresponsive gel photoreleasing nitric oxide for potential ocular applications.

We report herein the design, preparation, characterization and biological evaluation of a thermoresponsive gel based on binary mixtures of Pluronic® co-polymers F127 and P123, the latter being covalently functionalized with a nitric oxide (NO) photodonor (NOPD). The weight ratio between the two polymeric components is optimized in order to observe gelation of their saline water solution in the range of 32-35 °C, in order to exploit the therapeutic properties of NO for potential ocular applications. Rheological measurements were performed to evaluate the gelation temperature and, hence, to select a co-polymer mixture specifically appropriate for the reference application. Integration of the NOPD into the polymeric scaffold does not affect its rheological and spectroscopic properties, making it a good absorber of visible light both in solution and in the gel phase. Irradiation of the saline solution of the polymeric components with visible light triggers NO release, which occurs with an efficiency of more than one order of magnitude faster than that observed for the isolated NOPD. The polymeric system fully preserves such photobehavior after gelation as demonstrated by the effective NO photorelease from the gel matrix and its diffusion in the supernatant upon illumination. The gel is well-tolerated in both dark and light conditions by corneal cells, while being able to induce growth inhibition towards Staphylococcus aureus under visible light irradiation and has high moduli which can contribute to an adequate retention time within the eyes.

Contact Lenses Delivering Nitric Oxide under Daylight for Reduction of Bacterial Contamination.

Ocular infection due to microbial contamination is one of the main risks associated with the wearing of contact lens, which demands novel straightforward strategies to find reliable solutions. This contribution reports the preparation, characterization and biological evaluation of soft contact lenses (CL) releasing nitric oxide (NO), as an unconventional antibacterial agent, under daylight exposure. A tailored NO photodonor (NOPD) was embedded into commercial CL leading to doped CL with an excellent optical transparency (transmittance = 100%) at λ ≥ 450 nm. The NOPD results homogeneously distributed in the CL matrix where it fully preserves the photobehavior exhibited in solution. In particular, NO release from the CL and its diffusion in the supernatant physiological solution is observed upon visible light illumination. The presence of a blue fluorescent reporting functionality into the molecular skeleton of the NOPD, which activates concomitantly to the NO photorelease, allows the easy monitoring of the NO delivery in real-time and confirms that the doped CL work under daylight exposure. The NO photoreleasing CL are well-tolerated in both dark and light conditions by corneal cells while being able to induce good growth inhibition of Staphylococcus aureus under visible light irradiation. These results may pave the way to further engineering of the CL with NOPD as innovative ocular devices activatable by sunlight.

Visible light-activatable multicargo microemulsions with bimodal photobactericidal action and dual colour fluorescence.

In this contribution we report the design, preparation, and physico-chemical, photophysical and photochemical characterization of photoactivatable microemulsions (MEs) based on Labrasol®, isopropanol and Lauroglycol® FCC as a surfactant, co-surfactant and oily phase, respectively. The MEs co-incorporate, in their oil phase, two lipophilic guests such as a red emitting singlet oxygen (1O2) photosensitizer (PS) and a tailored green emitting nitric oxide (NO) photodonor (NOPD). These two chromofluorogenic units absorb in different spectral windows of the visible range, and their individual photophysical and photochemical properties are well-conserved when co-entrapped in the microemulsions. These features permit the PS and NOPD to operate either individually or in tandem resulting in (i) red, green or both fluorescence emission, (ii) photogeneration of cytotoxic 1O2, NO or both and (iii) amplified photobactericidal action against Staphylococcus aureus due to the combined effect of these two antibacterial agents.

Preclinical evidence of enhanced analgesic activity of duloxetine complexed with succinyl-ß-cyclodextrin: A comparative study with cyclodextrin complexes.

Chronic pain represents one of the most important public health problems, with a great prevalence of comorbidity with depression and cognitive decline. Antidepressants such as duloxetine, a serotonin-norepinephrine reuptake inhibitor, represent an essential part of the therapeutic strategy for chronic pain management in addition to classical analgesics. Duloxetine is endowed with good efficacy and a good profile of safety and tolerability. Yet, duloxetine is metabolized by the cytochrome P450 system 2D6 and 1A2 (CYP2D6 and CYP1A2) and it exhibits moderate inhibitory activity on CYP2D6, resulting in side effects and metabolic interactions that may occur on a long term therapeutic schedule. Cyclodextrins (CyDs) are used in pharmaceutical applications for numerous purposes, including the improvement of drug bioavailability. In order to evaluate their effects on the activity of duloxetine, we first spectrophotometrically studied the host-guest complexes obtained combining duloxetine and different ß-CyD derivatives (ß-CyD, ß-CyDen-c-(Glu-Glu), and succinyl-ß-CyD) and then performed in vivo and in vitro studies. Among duloxetine/CyDs complexes, succinyl-ß-CyD ameliorated the analgesic activity of duloxetine in the tail flick test and in the formalin test in mice and significantly protected the drug from CYP2D6 metabolism.

A phototherapeutic fluorescent ß-cyclodextrin branched polymer delivering nitric oxide.

We report herein on a novel water-soluble ß-cyclodextrin-branched polymer covalently integrating a fluorescein moiety and a nitric oxide (NO) photodonor within its macromolecular skeleton. Photoexcitation with visible light induces the parallel activation of the two chromophores, which results in the green fluorescence emission suitable for imaging accompanied by NO release for therapy. In fact, this polymer internalizes in squamous carcinoma cancer cells in vitro, visualized by fluorescence microscopy, and induces cell mortality as result of the NO photo-decaging. The non-covalent drug delivery capability of this new material is also demonstrated using a hydrophobic photosensitizer for photodynamic therapy as a probe.