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.

Red-Light-Photosensitized NO Release and Its Monitoring in Cancer Cells with Biodegradable Polymeric Nanoparticles.

The role of nitric oxide (NO) as an "unconventional" therapeutic and the strict dependence of biological effects on its concentration require the generation of NO with precise spatiotemporal control. The development of precursors and strategies to activate NO release by excitation in the so-called "therapeutic window" with highly biocompatible and tissue-penetrating red light is desirable and challenging. Herein, we demonstrate that one-photon red-light excitation of Verteporfin, a clinically approved photosensitizer (PS) for photodynamic therapy, activates NO release, in a catalytic fashion, from an otherwise blue-light activatable NO photodonor (NOPD) with an improvement of about 300 nm toward longer and more biocompatible wavelengths. Steady-state and time-resolved spectroscopic and photochemical studies combined with theoretical calculations account for an NO photorelease photosensitized by the lowest triplet state of the PS. In view of biological applications, the water-insoluble PS and NOPD have been co-entrapped within water-dispersible, biodegradable polymeric nanoparticles (NPs) of mPEG-b-PCL (about 84 nm in diameter), where the red-light activation of NO release takes place even more effectively than in an organic solvent solution and almost independently by the presence of oxygen. Moreover, the ideal spectroscopic prerequisites and the restricted environment of the NPs permit the green-fluorescent co-product formed concomitantly to NO photorelease to communicate with the PS via Förster resonance energy transfer. This leads to an enhancement of the typical red emission of the PS offering the possibility of a double color optical reporter useful for the real-time monitoring of the NO release through fluorescence techniques. The suitability of this strategy applied to the polymeric NPs as potential nanotherapeutics was evaluated through biological tests performed by using HepG2 hepatocarcinoma and A375 melanoma cancer cell lines. Fluorescence investigation in cells and cell viability experiments demonstrates the occurrence of the NO release under one-photon red-light illumination also in the biological environment. This confirms that the adopted 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 the use of sophisticated irradiation sources.

On the photobehaviour of curcumin in biocompatible hosts: The role of H-abstraction in the photodegradation and photosensitization.

Curcumin (CUR) is a naturally occurring pigment extensively studied due to its therapeutic activity and delivered by suitable nanocarriers to overcome poor solubility in aqueous media. The significant absorption of CUR in the visible blue region has prompted its use as a potential phototherapeutic agent in treating infectious and cancer diseases, although the mechanism underlying the phototoxic effects is still not fully understood. This contribution investigates the photobehaviour of CUR within polymeric micelles, microemulsions, and zein nanoparticles, chosen as biocompatible nanocarriers, and human serum albumin as a representative biomolecule. Spectroscopic studies indicate that in all host systems, the enolic tautomeric form of CUR is converted in a significant amount of the diketo form because of the perturbation of the intramolecular hydrogen bond. This leads to intermolecular H-abstraction from the host components by the lowest excited triplet state of CUR with the formation of the corresponding ketyl radical, detected by nanosecond laser flash photolysis. This radical is oxidized by molecular oxygen, likely generating peroxyl and hydroperoxyl radical species, unless in Zein, reasonably due to the poor availability of oxygen in the closely packed structure of this nanocarrier. In contrast, no detectable formation of singlet oxygen was revealed in all the systems. Overall these results highlight the key role of the H-abstraction process over singlet oxygen sensitization as a primary photochemical pathway strictly dictated by the specific features of the microenvironment, providing new insights into the photoreactivity of CUR in biocompatible hosts that can also be useful for a better understanding of its phototoxicity mechanism.

Different In Silico Approaches Using Heterocyclic Derivatives against the Binding between Different Lineages of SARS-CoV-2 and ACE2.

Over the last few years, the study of the SARS-CoV-2 spike protein and its mutations has become essential in understanding how it interacts with human host receptors. Since the crystallized structure of the spike protein bound to the angiotensin-converting enzyme 2 (ACE2) receptor was released (PDB code 6M0J), in silico studies have been performed to understand the interactions between these two proteins. Specifically, in this study, heterocyclic compounds with different chemical characteristics were examined to highlight the possibility of interaction with the spike protein and the disruption of the interaction between ACE2 and the spike protein. Our results showed that these compounds interacted with the spike protein and interposed in the interaction zone with ACE2. Although further studies are needed, this work points to these heterocyclic push-pull compounds as possible agents capable of interacting with the spike protein, with the potential for the inhibition of spike protein-ACE2 binding.

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.

Acidochromism of donor-acceptor Stenhouse adducts in organic solvent.

First generation DASA derivatives can be reversibly isomerized from the coloured, open form to the colourless, closed isomer upon protonation, thus behaving as acidochromic compounds in halogenated organic solvent.

A molecular dyad delivered by biodegradable polymeric nanoparticles for combined PDT and NO-PDT in cancer cells.

The design, synthesis, photochemical properties, and biological evaluation of a novel molecular dyad with double photodynamic action and its formulation within biodegradable polymeric nanoparticles (NPs) are reported. A BODIPY-based singlet oxygen (1O2) photosensitizer (PS) and a nitric oxide (NO) photodonor (NOPD) based on an amino-nitro-benzofurazan moiety have been covalently joined in a new molecular dyad, through a flexible alkyl spacer. Excitation of the dyad with visible light in the range 400-570 nm leads to the concomitant generation of the cytotoxic 1O2 and NO with effective quantum yields, being ΦΔ = 0.49 ± 0.05 and ΦNO = 0.18 ± 0.01, respectively. Besides, the non-fluorescent NOPD unit becomes highly fluorescent after the NO release, acting as an optical reporter for the NO photogenerated. The dyad is not soluble in water medium but can be effectively entrapped in water-dispersible, biodegradable polymeric NPs made of mPEG-PCL, ca. 66 nm in diameter. The polymeric nano-environment affects in an opposite way the photochemical performances of the dyad, reducing ΦΔ to 0.16 ± 0.02 and increasing ΦNO to 0.92 ± 0.03, respectively. The NPs effectively deliver the photoactive cargo into the cytoplasm of HepG2 hepatocellular carcinoma cells. A remarkable level of cell mortality is observed for the loaded NPs at very low concentrations of the dyad (1-5 µM) and very low light doses (≤0.8 J cm-2) more likely as the result of the combined photodynamic action of 1O2 and NO.

Doxorubicin-NO Releaser Molecular Hybrid Activatable by Green Light to Overcome Resistance in Breast Cancer Cells.

The biological activity of a molecular hybrid (DXNO-GR) joining doxorubicin (DOX) and an N-nitroso moiety releasing nitric oxide (NO) under irradiation with the biocompatible green light has been investigated against DOX-sensitive (MCF7) and -resistant (MDA-MB-231) breast cancer cells in vitro. DXNO-GR shows significantly higher cellular internalization than DOX in both cell lines and, in contrast to DOX, does not experience cell efflux in MDR overexpressing MDA-MB-231 cells. The higher cellular internalization of the DXNO-GR hybrid seems to be mediated by bovine serum albumin (BSA) as a suitable carrier among serum proteins, according to the high binding constant measured for DXNO-GR, which is more than one order of magnitude larger than that reported for DOX. Despite the higher cellular accumulation, DXNO-GR is not toxic in the dark but induces remarkable cell death following photoactivation with green light. This lack of dark toxicity is strictly related to the different cellular compartmentalization of the molecular hybrid that, different from DOX, does not localize in the nucleus but is mainly confined in the Golgi apparatus and endoplasmic reticulum and therefore does not act as a DNA intercalator. The photochemical properties of the hybrid are not affected by binding to BSA as demonstrated by the direct detection of NO photorelease, suggesting that the reduction of cell viability observed under light irradiation is a combined effect of DOX phototoxicity and NO release which, ultimately, inhibits MDR1 efflux pump in DOX-resistant cells.

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.

A Supramolecular Nanoassembly of Lenvatinib and a Green Light-Activatable NO Releaser for Combined Chemo-Phototherapy.

The chemotherapeutic Lenvatinib (LVB) and a nitric oxide (NO) photodonor based on a rhodamine antenna (RD-NO) activatable by the highly compatible green light are supramolecularly assembled by a ß-cyclodextrin branched polymer (PolyCD). The poorly water-soluble LVB and RD-NO solubilize very well within the polymeric host leading to a ternary supramolecular nanoassembly with a diameter of ~55 nm. The efficiency of the NO photorelease and the typical red fluorescence of RD-NO significantly enhance within the polymer due to its active role in the photochemical and photophysical deactivation pathways. The co-presence of LVB within the same host does not affect either the nature or the efficiency of the photoinduced processes of RD-NO. Besides, irradiation of RD-NO does not lead to the decomposition of LVB, ruling out any intermolecular photoinduced process between the two guests despite sharing the same host. Ad-hoc devised Förster Resonance Energy Transfer experiments demonstrate this to be the result of the not close proximity of the two guests, which are confined in different compartments of the same polymeric host. The supramolecular complex is stable in a culture medium, and its biological activity has been evaluated against HEP-G2 hepatocarcinoma cell lines in the dark and under irradiation with visible green light, using LVB at a concentration well below the IC50. Comparative experiments performed using the polymeric host encapsulating the individual LVB and RD-NO components under the same experimental conditions show that the moderate cell mortality induced by the ternary complex in the dark increases significantly upon irradiation with visible green light, more likely as the result of synergism between the NO photogenerated and the chemotherapeutic.

Phosphonodithioester-amine coupling in water: a fast reaction to modify the surface of liposomes.

The incorporation of lipophilic phosphonodithioesters in phospholipid formulations generates clickable liposomes that react with amines. The kinetics of this metal free phosphonodithioester-amine coupling (PAC) on liposomes in water is reported and can be classified as a fast reaction with a second order rate constant of k ≈ 8 × 102 M-1 s-1. The PAC reaction represents a versatile strategy to functionalize liposomes.

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 generator of peroxynitrite activatable with red light.

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as "unconventional" therapeutics with precise spatiotemporal control by using light stimuli may open entirely new horizons for innovative therapeutic modalities. Among ROS and RNS, peroxynitrite (ONOO-) plays a dominant role in chemistry and biology in view of its potent oxidizing power and cytotoxic action. We have designed and synthesized a molecular hybrid based on benzophenothiazine as a red light-harvesting antenna joined to an N-nitroso appendage through a flexible spacer. Single photon red light excitation of this molecular construct triggers the release of nitric oxide (˙NO) and simultaneously produces superoxide anions (O2˙-). The diffusion-controlled reaction between these two radical species generates ONOO-, as confirmed by the use of fluorescein-boronate as a highly selective chemical probe. Besides, the red fluorescence of the hybrid allows its tracking in different types of cancer cells where it is well-tolerated in the dark but induces remarkable cell mortality under irradiation with red light in a very low concentration range, with very low light doses (ca. 1 J cm-2). This ONOO- generator activatable by highly biocompatible and tissue penetrating single photon red light can open up intriguing prospects in biomedical research, where precise and spatiotemporally controlled concentrations of ONOO- are required.

Development of Spirulina sea-weed raw extract/polyamidoamine hydrogel system as novel platform in photodynamic therapy: Photostability and photoactivity of chlorophyll a.

The aim of this paper is to present and characterize Polyamidoamine-based hydrogels (PAA) as scaffolds to host photoactive Chlorophyll a (Chl a) from Spirulina (Arthrospira platensis) sea-weed Extract (SE), for potential applications in Photodynamic Therapy (PDT). The pigment extracted from SE was blended inside PAA without further purification, according to Green Chemistry principles. A comprehensive investigation of this hybrid platform, PAA/SE-based, was thus performed in our laboratory and, by means of Visible absorption and emission spectroscopies, the Chl a features, stability and photoactivity were studied. The obtained results evidenced the presence of two main Chl a forms, monomeric and dimeric, interacting with hydrogel polyamidoamines network. To better understand the nature of this interaction, the spectroscopic investigation of this system was performed both before and after the solidification of the hydrogel, that occurred at least in 24 h. Then, focusing the attention on solid scaffold, the 1Chl a⁎ fluorescence lifetime and FTIR-ATR analyses of PAA/SE were carried out, confirming the findings. The swelling and Point Zero Charge (PZC) measurements of solid PAA and PAA/SE were additionally performed to investigate the hydrogel behavior in water. Chl a molecules blended in PAA were (photo) stable and photoactive, and this latter feature was demonstrated showing that the pigment induced, when swelled in water and under irradiation, the formation of singlet oxygen (1O2), measured by direct and indirect methods.

NO release regulated by doxorubicin as the green light-harvesting antenna.

We report for the first time a NO photodonor (NOPD) operating with the widely used chemotherapeutic agent doxorubicin (DOX) as the light-harvesting antenna. This permits NO uncaging from an N-nitroso appendage upon selective excitation of DOX with highly biocompatible green light, without precluding its typical red emission. This NOPD effectively binds DNA and photodelivers NO nearby, representing an intriguing candidate for potential multimodal therapeutic applications based on the combination of DOX and NO.

DNA-Targeted NO Release Photoregulated by Green Light.

A novel molecular hybrid has been designed and synthesized in which acridine orange (AO) is covalently linked to an N-nitrosoaniline derivative through an alkyl spacer. Photoexcitation of the AO antenna with the highly biocompatible green light results in intense fluorescence emission and triggers NO detachment from the N-nitroso appendage via an intramolecular electron transfer. The presence of the AO moiety encourages the binding with DNA through both external and partially intercalative fashions, depending on the DNA:molecular hybrid molar ratio. Importantly, this dual-mode binding interaction with the biopolymer does not preclude the NO photoreleasing performances of the molecular hybrid, permitting NO to be photogenerated nearby DNA with an efficiency similar to that of the free molecule. These properties make the presented compound an intriguing candidate for fundamental and potential applicative research studies where NO delivery in the DNA proximity precisely regulated by harmless green light is required.

Photodegradation of Antibiotics by Noncovalent Porphyrin-Functionalized TiO2 in Water for the Bacterial Antibiotic Resistance Risk Management.

Antibiotics represent essential drugs to contrast the insurgence of bacterial infections in humans and animals. Their extensive use in livestock farming, including aquaculture, has improved production performances and food safety. However, their overuse can implicate a risk of water pollution and related antimicrobial resistance. Consequently, innovative strategies for successfully removing antibiotic contaminants have to be advanced to protect human health. Among them, photodegradation TiO2-driven under solar irradiation appears not only as a promising method, but also a sustainable pathway. Hence, we evaluated several composite TiO2 powders with H2TCPP, CuTCPP, ZnTCPP, and SnT4 porphyrin for this scope in order to explore the effect of porphyrins sensitization on titanium dioxide. The synthesis was realized through a fully non-covalent functionalization in water at room conditions. The efficacy of obtained composite materials was also tested in photodegrading oxolinic acid and oxytetracycline in aqueous solution at micromolar concentrations. Under simulated solar irradiation, TiO2 functionalized with CuTCPP has shown encouraging results in the removal of oxytetracycline from water, by opening the way as new approaches to struggle against antibiotic's pollution and, finally, to represent a new valuable tool of public health.

Overcoming Doxorubicin Resistance with Lipid-Polymer Hybrid Nanoparticles Photoreleasing Nitric Oxide.

We report on tailored lipid-polymer hybrid nanoparticles (NPs) delivering nitric oxide (NO) under the control of visible light as a tool for overcoming doxorubicin (DOX) resistance. The NPs consist of a polymeric core and a coating. They are appropriately designed to entrap DOX in the poly(lactide-co-glycolide) core and a NO photodonor (NOPD) in the phospholipid shell to avoid their mutual interaction both in the ground and excited states. The characteristic red fluorescence of DOX, useful for its tracking in cells, is well preserved upon incorporation within the NPs, even in the copresence of NOPD. The NP scaffold enhances the NO photoreleasing efficiency of the entrapped NOPD when compared with that of the free compound, and the copresence of DOX does not significantly affect such enhanced photochemical performance. Besides, the delivery of DOX and NOPD from NPs is also not mutually influenced. Experiments carried out in M14 DOX-resistant melanoma cells demonstrate that NO release from the multicargo NPs can be finely regulated by excitation with visible light, at a concentration level below the cytotoxic doses but sufficient enough to inhibit the efflux transporters mostly responsible for DOX cellular extrusion. This results in increased cellular retention of DOX with consequent enhancement of its antitumor activity. This approach, in principle, is not dependent on the type of chemotherapeutic used and may pave the way for new treatment modalities based on the photoregulated release of NO to overcome the multidrug resistance phenomenon and improve cancer chemotherapies.

Enhancing doxorubicin anticancer activity with a novel polymeric platform photoreleasing nitric oxide.

Combinations of conventional chemotherapeutics with unconventional anticancer agents such as reactive oxygen and nitrogen species may offer treatment benefits for cancer therapies. Here we report a novel polymeric platform combining the delivery of Doxorubicin (DOXO) with the light-regulated release of nitric oxide (NO). An amphiphilic block-copolymer (P1) was designed and synthesized as the drug carrier, with pendant amine groups to attach DOXO via a urea linkage and a NO photodonor (NOPD) activable by visible light. The two grafted-copolymers (P1-DOXO and P1-NOPD) self-assembled via solvent displacement methods into nanoparticles (NPs), containing both therapeutic components (NP1) and, for comparison, the individual NOPD (NP2) and DOXO (NP3). All the NPs were fully characterized in terms of physicochemical, photochemical and photophysical properties. These experiments demonstrated that integration of the NOPD within the polymeric scaffold enhanced the NO photoreleasing efficiency when compared with the free NOPD, and that the proximity to DOXO on the polymer chains did not significantly affect the enhanced photochemical performance. Internalization of the NPs into lung, intestine, and skin cancer cell lines was investigated after co-formulation with Cy5 fluorescent tagged polymers, and cytotoxicity of the NPs against the same panel of cell lines was assessed under dark and light conditions. The overall results demonstrate effective cell internalization of the NPs and a notable enhancement in killing activity of the dual-action therapeutic NP1 when compared with NP2, NP3 and the free DOXO, respectively. This suggests that the combination of DOXO with photoregulated NO release, achieved through the mixed formulation strategy of tailored polymer conjugate NPs, may open new treatment modalities based on the use of NO to improve cancer therapies.

Combination of PDT and NOPDT with a Tailored BODIPY Derivative.

The engineering of photosensitizers (PS) for photodynamic therapy (PDT) with nitric oxide (NO) photodonors (NOPD) is broadening the horizons for new and yet to be fully explored unconventional anticancer treatment modalities that are entirely controlled by light stimuli. In this work, we report a tailored boron-dipyrromethene (BODIPY) derivative that acts as a PS and a NOPD simultaneously upon single photon excitation with highly biocompatible green light. The photogeneration of the two key species for PDT and NOPDT, singlet oxygen (1O2) and NO, has been demonstrated by their direct detection, while the formation of NO is shown not to be dependent on the presence of oxygen. Biological studies carried out using A375 and SKMEL28 cancer cell lines, with the aid of suitable model compounds that are based on the same BODIPY light harvesting core, unambiguously reveal the combined action of 1O2 and NO in inducing amplified cancer cell mortality exclusively under irradiation with visible green light.