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Anna Piperno,1 Maria Teresa Sciortino,1 Elena Giusto,2 Monica Montesi,2 Silvia Panseri,2 Angela Scala1 1Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; 2CNR-ISTEC, Institute of Science and Technology for Ceramics, National Research Council of Italy, Faenza, RA, ItalyCorrespondence: Angela ScalaDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, ItalyTel +39 090 6765515Fax +39 090 393897Email ascala@unime.itAbstract: Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly‐interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.Keywords: PLGA, PLA, siRNA, CRISPR, COVID-19, CLAN

Increased global research is focused on the development of novel therapeutics to combat antimicrobial and antiviral resistance. Pistachio nuts represent a good source of protein, fiber, monounsaturated fatty acids, minerals, vitamins, and phytochemicals (carotenoids, phenolic acids, flavonoids and anthocyanins). The phytochemicals found in pistachios are structurally diverse compounds with antimicrobial and antiviral potential, demonstrated as individual compounds, extracts and complexed into nanoparticles. Synergistic effects have also been reported in combination with existing drugs. Here we report an overview of the antimicrobial and antiviral potential of pistachio nuts: studies show that Gram-positive bacterial strains, such as Staphylococcus aureus , are the most susceptible amongst bacteria, whereas antiviral effect has been reported against herpes simplex virus 1 (HSV-1). Amongst the known pistachio compounds, zeaxanthin has been shown to affect both HSV-1 attachment penetration of human cells and viral DNA synthesis. These data suggest that pistachio extracts and derivatives could be used for the topical treatment of S. aureus skin infections and ocular herpes infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mandalari, Pennisi, Gervasi and Sciortino.)

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CL pro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CL pro and PL pro , but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

Epstein-Barr (EBV) is a human γ-herpesvirus that undergoes both a productive (lytic) cycle and a non-productive (latent) phase. The virus establishes enduring latent infection in B lymphocytes and productive infection in the oral mucosal epithelium. Like other herpesviruses, EBV expresses its genes in a coordinated pattern during acute infection. Unlike others, it replicates its DNA during latency to maintain the viral genome in an expanding pool of B lymphocytes, which are stimulated to divide upon infection. The reactivation from the latent state is associated with a productive gene expression pattern mediated by virus-encoded transcriptional activators BZLF-1 and BRLF-1. EBV is a highly transforming virus that contributes to the development of human lymphomas. Though viral vectors and mRNA platforms have been used to develop an EBV prophylactic vaccine, currently, there are no vaccines or antiviral drugs for the prophylaxis or treatment of EBV infection and EBV-associated cancers. Natural products and bioactive compounds are widely studied for their antiviral potential and capability to modulate intracellular signaling pathways. This review was intended to collect information on plant-derived products showing their antiviral activity against EBV and evaluate their feasibility as an alternative or adjuvant therapy against EBV infections and correlated oncogenesis in humans.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CL pro (also known as the main protease or M pro ) and PL pro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CL pro and PL pro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7- O -glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PL pro and 3CL pro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CL pro and PL pro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

The activation of the innate immune response during HSV-1 infection stimulates several transcription factors, such as NF-κB and IRF3, which are critical regulators of IFN-β expression. The released IFN-β activates the ISGs, which encode antiviral effectors such as the PKR. We found that HSV-1 triggers an antiviral transcriptional response during viral replication by activating TBK1-IRF3-NF-κB network kinetically. In contrast, we reported that infected PKR -/- cells fail to activate the transcription of TBK1. Downstream, TBK1 was unable to activate the transcription of IRF3 and NF-κB. These data suggested that in PKR -/- cells, HSV-1 replication counteracts TBK1-IRF3-NF-κB network. In this scenario, a combined approach of gene knockout and gene silencing was used to determine how the lack of PKR facilitates HSV-1 replication. We reported that in HEp-2-infected cells, PKR can influence the TBK1-IRF3-NF-κB network, consequently interfering with viral replication. Otherwise, an abrogated PKR-mediated signaling sustains the HSV-1 replication. Our result allows us to add additional information on the complex HSV-host interaction network by reinforcing the concept of the PKR role in the innate response-related networks during HSV replication in an in vitro model.

The search for alternative clinical treatments to fight resistance and find alternative antiviral treatments for the herpes simplex virus (HSV) is of great interest. Plants are rich sources of novel antiviral, pharmacologically active agents that provide several advantages, including reduced side effects, less resistance, low toxicity, and different mechanisms of action. In the present work, the antiviral activity of Californian natural raw (NRRE) and roasted unsalted (RURE) pistachio polyphenols-rich extracts was evaluated against HSV-1 using VERO cells. Two different extraction methods, with or without n -hexane, were used. Results showed that n -hexane-extracted NRRE and RURE exerted an antiviral effect against HSV-1, blocking virus binding on the cell surface, affecting viral DNA synthesis as well as accumulation of ICP0, UL42, and Us11 viral proteins. Additionally, the identification and quantification of phenolic compounds by RP-HPLC-DAD confirmed that extraction with n -hexane exclusively accumulated tocopherols, carotenoids, and xanthophylls. Amongst these, zeaxanthin exhibited strong antiviral activity against HSV-1 (CC 50 : 16.1 µM, EC 50 4.08 µM, SI 3.96), affecting both the viral attachment and penetration and viral DNA synthesis. Zeaxanthin is a dietary carotenoid that accumulates in the retina as a macular pigment. The use of pistachio extracts and derivates should be encouraged for the topical treatment of ocular herpetic infections.

The almond industry produces, by bleaching and stripping, two by-products: blanched skin (BS) and blanch water (BW). The aim of this study was to investigate the nutritional and polyphenolic profile, as well as the antioxidant, antimicrobial, antiviral, and potential prebiotic effects of BS and BW from three different Sicilian cultivars. The total phenols and flavonoids contents were ≥1.72 and ≥0.56 g gallic acid equivalents and ≥0.52 and ≥0.18 g rutin equivalents/100 g dry extract (DE) in BS and BW, respectively. The antioxidant activity, evaluated by 2,2-diphenyl-1-picrylhydrazyl scavenging ability, trolox equivalent antioxidant capacity, ferric-reducing antioxidant power, and oxygen radical absorbance capacity, was ≥3.07 and ≥0.83 g trolox equivalent/100 g DE in BS and BW, respectively. Isorhamnetin-3- O -glucoside was the most abundant flavonoid detected in both by-products. No antimicrobial effect was recorded, whereas BS samples exerted antiviral activity against herpes simplex virus 1 (EC 50 160.96 μg/mL). BS also showed high fibre (≥52.67%) and protein (≥10.99) contents and low fat (≤15.35%) and sugars (≤5.55%), making it nutritionally interesting. The present study proved that the cultivar is not a discriminating factor in determining the chemical and biological properties of BS and BW.

The present study aims to assess the antioxidant and antiviral effectiveness of leaf extracts obtained from Olea europaea L. var. sativa and Olea europaea L. var. sylvestris . The total antioxidant activity was determined via both an ammonium phosphomolybdate assay and a nitric oxide radical inhibition assay. Both extracts showed reducing abilities in an in vitro system and in human HeLa cells. Indeed, after oxidative stress induction, we found that exposition to olive leaf extracts protects human HeLa cells from lipid peroxidation and increases the concentration of enzyme antioxidants such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase. Additionally, OESA treatment affects viral DNA accumulation more than OESY, probably due to the exclusive oleuropein content. In fact, subtoxic concentrations of oleuropein inhibit HSV-1 replication, stimulating the phosphorylation of PKR, c-FOS, and c-JUN proteins. These results provide new knowledge about the potential health benefits and mechanisms of action of oleuropein and oleuropein-rich extracts.

Punica granatum is a rich source of bioactive compounds which exhibit various biological effects. In this study, pomegranate peel and leaf ethanolic crude extracts (PPE and PLE, respectively) were phytochemically characterized and screened for antioxidant, antimicrobial and antiviral activity. LC-PDA-ESI-MS analysis led to the identification of different compounds, including ellagitannins, flavonoids and phenolic acids. The low IC 50 values, obtained by DPPH and FRAP assays, showed a noticeable antioxidant effect of PPE and PLE comparable to the reference standards. Both crude extracts and their main compounds (gallic acid, ellagic acid and punicalagin) were not toxic on Vero cells and exhibited a remarkable inhibitory effect on herpes simplex type 1 (HSV-1) viral plaques formation. Specifically, PPE inhibited HSV-1 adsorption to the cell surface more than PLE. Indeed, the viral DNA accumulation, the transcription of viral genes and the expression of viral proteins were significantly affected by PPE treatment. Amongst the compounds, punicalagin, which is abundant in PPE crude extract, inhibited HSV-1 replication, reducing viral DNA and transcripts accumulation, as well as proteins of all three phases of the viral replication cascade. In contrast, no antibacterial activity was detected. In conclusion, our findings indicate that Punica granatum peel and leaf extracts, especially punicalagin, could be a promising therapeutic candidate against HSV-1.

The HSV-1 tegument protein Us11 counteracts the antiviral defense mechanisms by precluding the host protein shutoff. Previous works demonstrated that Us11 prevents heat-and staurosporine-induced apoptosis and inhibits autophagy. Therefore, in the present study, we investigated the hypothesis that HSV-1, through Us11, could recruit caspase-8, a key enzyme regulating programmed cell death. We first show that HSV-1 promotes the accumulation of caspase-8-p18 active fragments in both semi permissive THP-1 cells and fully permissive HEp-2 cells to HSV-1 replication. Using a recombinant virus R3630 (ΔUs11/ΔUs12) and a plasmid encoding Us11-recombinant protein we have proven that Us11 promotes p18 accumulation, which does not trigger the apoptotic signaling. Additional, in an in vitro model, we demonstrated that Us11-recombinant protein induces caspase-8-p18 cleavage by physically interacting with the caspase-8 recombinant protein. Finally, we found that, during HSV-1 replication, activated-caspase-8 cleaves Atg3 protein to potentially block autophagy and support its replication., (© 2022. The Author(s).)

In the last decade, nanotechnological progress has generated new opportunities to improve the safety and efficacy of conventional anticancer therapies. Compared with other carriers, graphene nanoplatforms possess numerous tunable functionalities for the loading of multiple bioactive compounds, although their biocompatibility is still a debated concern. Recently, we have investigated the modulation of genes involved in cancer-associated canonical pathways induced by graphene engineered with cyclodextrins (GCD). Here, we investigated the GCD impact on cells safety, the HEp-2 responsiveness to Doxorubicin (DOX) and the cancer-related intracellular signalling pathways modulated by over time exposure to DOX loaded on GCD (GCD@DOX). Our studies evidenced that both DOX and GCD@DOX induced p53 and p21 signalling resulting in G 0 /G 1 cell cycle arrest. A genotoxic behaviour of DOX was reported via detection of CDK (T14/Y15) activation and reduction of Wee-1 expression. Similarly, we found a cleavage of PARP by DOX within 72 h of exposure. Conversely, GCD@DOX induced a late cleavage of PARP, which could be indicative of less toxic effect due to controlled release of the drug from the GCD nanocarrier. Finally, the induction of the autophagy process supports the potential recycling of DOX with the consequent limitation of its toxic effects. Together, these findings demonstrate that GCD@DOX is a biocompatible drug delivery system able to evade chemoresistance and doxorubicin toxicity.

Olea europaea L. var. sativa (OESA) preparations are widely used in traditional medicine in the Mediterranean region to prevent and treat different diseases. In this research, olive extracts derived from the leaves of the OESA tree have been screened for antioxidant activity by two methods: the DPPH free radical scavenging assay (DPPH) and the Ferric reducing antioxidant power (FRAP) assay. The DPPH assay showed that OESA possesses a stronger antioxidant activity (84%) at 1 mg/mL while the FRAP method showed a strong metal ion chelating activity (90%) at 1 mg/mL. The low IC 50 values, obtained by two different methods, implies that OESA has a noticeable effect on scavenging free radicals comparable to standards. During EBV infection, the free radicals increased triggering lipid oxidation. Therefore, the monitoring of the secondary lipid peroxidation products was done by measuring malonaldehyde (MDA) and conjugated dienes (DC). The simultaneous treatment of Raji cells with OESA and TPA, as an inductorof the lytic cycle, generated a significant decrease in MDA levels and DC ( p < 0.05). Besides, Raji cells simultaneously exposed to TPA and OESA exhibited a percentage of EBV-positive fluorescence cells lower than TPA treated cells (**** p < 0.0001). This suggests that OESA treatment has a protective effect against EBV lytic cycle induction.

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 μg/mL and between 15.61 and 31.25 μg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC 50 ) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.

Viral infections are responsible for several chronic and acute diseases in both humans and animals. Despite the incredible progress in human medicine, several viral diseases, such as acquired immunodeficiency syndrome, respiratory syndromes, and hepatitis, are still associated with high morbidity and mortality rates in humans. Natural products from plants or other organisms are a rich source of structurally novel chemical compounds including antivirals. Indeed, in traditional medicine, many pathological conditions have been treated using plant-derived medicines. Thus, the identification of novel alternative antiviral agents is of critical importance. In this review, we summarize novel phytochemicals with antiviral activity against human viruses and their potential application in treating or preventing viral disease.

The synthesis of α-fluorinated methyl ketones has always been challenging. New methods based on the homologation chemistry via nucleophilic halocarbenoid transfer, carried out recently in our labs, allowed us to design and synthesize a target-directed dipeptidyl α,α-difluoromethyl ketone (DFMK) 8 as a potential antiviral agent with activity against human coronaviruses. The ability of the newly synthesized compound to inhibit viral replication was evaluated by a viral cytopathic effect (CPE)-based assay performed on MCR5 cells infected with one of the four human coronaviruses associated with respiratory distress, i.e., hCoV-229E, showing antiproliferative activity in the micromolar range (EC 50 = 12.9 ± 1.22 µM), with a very low cytotoxicity profile (CC 50 = 170 ± 3.79 µM, 307 ± 11.63 µM, and 174 ± 7.6 µM for A549, human embryonic lung fibroblasts (HELFs), and MRC5 cells, respectively). Docking and molecular dynamics simulations studies indicated that 8 efficaciously binds to the intended target hCoV-229E main protease (M pro ). Moreover, due to the high similarity between hCoV-229E M pro and SARS-CoV-2 M pro , we also performed the in silico analysis towards the second target, which showed results comparable to those obtained for hCoV-229E M pro and promising in terms of energy of binding and docking pose.

The graphene road in nanomedicine still seems very long and winding because the current knowledge about graphene/cell interactions and the safety issues are not yet sufficiently clarified. Specifically, the impact of graphene exposure on gene expression is a largely unexplored concern. Herein, we investigated the intracellular fate of graphene (G) decorated with cyclodextrins (CD) and loaded with doxorubicin (DOX) and the modulation of genes involved in cancer-associated canonical pathways. Intracellular fate of GCD@DOX, tracked by FLIM, Raman mapping and fluorescence microscopy, evidenced the efficient cellular uptake of GCD@DOX and the presence of DOX in the nucleus, without graphene carrier. The NanoString nCounter™ platform provided evidence for 34 (out of 700) differentially expressed cancer-related genes in HEp-2 cells treated with GCD@DOX (25 µg/mL) compared with untreated cells. Cells treated with GCD alone (25 µg/mL) showed modification for 16 genes. Overall, 14 common genes were differentially expressed in both GCD and GCD@DOX treated cells and 4 of these genes with an opposite trend. The modification of cancer related genes also at sub-cytotoxic G concentration should be taken in consideration for the rational design of safe and effective G-based drug/gene delivery systems. The reliable advantages provided by NanoString ® technology, such as sensibility and the direct RNA measurements, could be the cornerstone in this field.

Graphene-based materials are intriguing nanomaterials with applications ranging from nanotechnology-related devices to drug delivery systems and biosensing. Multifunctional graphene platforms were proposed for the detection of several typical biomarkers (i.e., circulating tumor cells, exosomes, circulating nucleic acids, etc.) in liquid biopsy, and numerous methods, including optical, electrochemical, surface-enhanced Raman scattering (SERS), etc., have been developed for their detection. Due to the massive advancements in biology, material chemistry, and analytical technology, it is necessary to review the progress in this field from both medical and chemical sides. Liquid biopsy is considered a revolutionary technique that is opening unexpected perspectives in the early diagnosis and, in therapy monitoring, severe diseases, including cancer, metabolic syndrome, autoimmune, and neurodegenerative disorders. Although nanotechnology based on graphene has been poorly applied for the rapid diagnosis of viral diseases, the extraordinary properties of graphene (i.e., high electronic conductivity, large specific area, and surface functionalization) can be also exploited for the diagnosis of emerging viral diseases, such as the coronavirus disease 2019 (COVID-19). This review aimed to provide a comprehensive and in-depth summarization of the contribution of graphene-based nanomaterials in liquid biopsy, discussing the remaining challenges and the future trend; moreover, the paper gave the first look at the potentiality of graphene in COVID-19 diagnosis., Competing Interests: The authors declare no conflict of interest

The current emergency due to the worldwide spread of the COVID-19 caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a great concern for global public health. Already in the past, the outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle Eastern respiratory syndrome (MERS) in 2012 demonstrates the potential of coronaviruses to cross-species borders and further underlines the importance of identifying new-targeted drugs. An ideal antiviral agent should target essential proteins involved in the lifecycle of SARS-CoV. Currently, some HIV protease inhibitors (i.e., Lopinavir) are proposed for the treatment of COVID-19, although their effectiveness has not yet been assessed. The main protease (M pr ) provides a highly validated pharmacological target for the discovery and design of inhibitors. We identified potent M pr inhibitors employing computational techniques that entail the screening of a Marine Natural Product (MNP) library. MNP library was screened by a hyphenated pharmacophore model, and molecular docking approaches. Molecular dynamics and re-docking further confirmed the results obtained by structure-based techniques and allowed this study to highlight some crucial aspects. Seventeen potential SARS-CoV-2 M pr inhibitors have been identified among the natural substances of marine origin. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds could be bioactive is excellent.

To replicate, spread and persist in the host environment, viruses have evolved several immunological escape mechanisms via the action of specific viral proteins. The model "host shut off" adopted by virion host shut off (VHS) protein of Herpes simplex type 1 (HSV-1) represents an immune evasion mechanism which affects the best-characterized component of the innate immunological response, protein kinase R (PKR). However, up to now, the real mechanism employed by VHS to control PKR is still unknown. In this paper, we implement and extend our previous findings reporting that wild-type HSV-1 is able to control PKR, whereas a VHS mutant virus (R2621) clearly induces an accumulation of phosphorylated PKR in several cell types in a VHS-RNase activity-dependent manner. Furthermore, we demonstrate for the first time a new PKR-regulatory mechanism based on the involvement of Us3 and UL13 tegument viral proteins. The combined approach of transfection and infection assay was useful to discover the new role of both viral proteins in the immunological escape and demonstrate that Us3 and UL13 control the accumulation of the phosphorylated form (ph-PKR). Lastly, since protein kinases are tightly regulated by phosphorylation events and, at the same time, phosphorylate other proteins by inducing post-translational modifications, the interplay between Us3 and VHS during HSV-1 infection has been investigated. Interestingly, we found that VHS protein accumulates at higher molecular weight following Us3 transfection, suggesting an Us3-mediated phosphorylation of VHS. These findings reveal a new intriguing interplay between viral proteins during HSV-1 infection involved in the regulation of the PKR-mediated immune response.

Natural compounds are a prominent source of novel antiviral drugs. Several reports have previously shown the antimicrobial activity of pistachio polyphenol extracts. Therefore, the aim of our research was to investigate the activity of polyphenol-rich extracts of natural shelled (NPRE) pistachios kernels ( Pistacia vera L.) on herpes simplex virus type 1 (HSV-1) replication. The Vero cell line was used to assess the cytotoxicity and antiviral activity. The cell viability was calculated by detection of cellular ATP after treatment with various concentrations of NPRE. For antiviral studies, five nontoxic-concentrations (0.1, 0.2, 0.4, 0.6, 0.8 mg/mL) were tested. Our study demonstrated that treatment with NPRE (0.4, 0.6, 0.8 mg/mL) reduced the expression of the viral proteins ICP8 (infected cell polypeptide 8), UL42 (unique long UL42 DNA polymerase processivity factor) , and US11 (unique short US11 protein), and resulted in a decrease of viral DNA synthesis. The 50% cytotoxic concentration (CC 50 ), 50% inhibitory concentration (EC 50 ), and the selectivity index (SI) values for NPRE were 1.2 mg/mL, 0.4mg/mL, and 3, respectively. Furthermore, we assessed the anti-herpetic effect of a mix of pure polyphenol compounds (NS MIX) present in NPRE. In conclusion, our findings indicate that natural shelled pistachio kernels have remarkable inhibitory activity against HSV-1., Competing Interests: The authors declare no conflict of interest.

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles <200 nm), despite the fact that the GCD@Ada-Rhod size is about 400 nm (by DLS and TEM data). We supposed that the internalization pathway was driven by physical-chemical features of GCD@Ada-Rhod, and the caveolae-mediated uptake enhanced the transfection efficiency, avoiding the lysosomal acid degradation. The cellular effects of internalized miRNA-15a on the oncogene protein BCL-2 were investigated at two different concentrations (N/P = 10 and 5), and a reduction of the BCL-2 level was detected at a low concentration (i.e., N/P = 10). miRNA-15a is considered an ideal cancer therapy molecule due to its activity on multiple transcription factors, and the elucidation of the correlation between the concentration of delivered miRNA-15a and the down-/up-regulation of the BCL-2 level, documented for the first time in this work, could be an important contribution to guide its clinical application.

Due to their antimicrobial and antiviral activity potential in vitro, polyphenols are gaining a lot of attention from the pharmaceutical and healthcare industries. A novel antiviral and antimicrobial approach could be based on the use of polyphenols obtained from natural sources. Here, we tested the antibacterial and antiviral effect of a mix of polyphenols present in natural almond skin (NS MIX). The antimicrobial potential was evaluated against the standard American Type Culture Collection (ATCC) and clinical strains of Staphylococcus aureus , including methicillin-resistant (MRSA) strains, by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Herpes simplex virus type I was used for the antiviral assessment of NS MIX by plaque assay. Furthermore, we evaluated the expression of viral cascade antigens. NS MIX exhibited antimicrobial (MIC values of 0.31-1.25 mg/ml) and antiviral activity (decrease in the viral titer ** p < 0.01, and viral DNA accumulation * p < 0.05) against Staphylococcus aureus and HSV-1, respectively. Amongst the isolated compounds, the aglycones epicatechin and catechin showed the greatest activity against S. aureus ATCC 6538P (MIC values of 0.078-0.15 and 0.15 mg/ml, respectively), but were not active against all the other strains. These results could be used to develop novel products for topical use.

Drug performance in the gastrointestinal tract (GIT) plays a crucial role in determining release and absorption. In the present work, we assessed the in vitro digestion of two synthetic N1-aryl-2-arylthioacetamidobenzimidazoles (NAABs), NAAB-496 and NAAB-503, using bio-relevant models of the human stomach and small intestine. The activity of NAAB-496 and NAAB-503 against herpes simplex virus (HSV-1) replication was also investigated. NAAB-496 was resistant to pepsin in the gastric environment, with a virtual 100% recovery, which decreased to 43.2% in the small intestine. NAAB-503 was sensitive to pepsin, with 65.7% degradation after 120 min gastric phase. 1H Nuclear magnetic resonance (NMR) post in vitro digestion highlighted an alteration of NAAB-496 after the gastric phase, whereas NAAB-503 appeared comparable to the original spectral data. Both NAAB-496 and NAAB-503 revealed some antiviral activity anti-HSV-1. The 50% effective concentration (EC50) of the compounds was 0.058 mg/mL for NAAB-496 and 0.066 for NAAB-503. Future studies will evaluate the behavior of NAAB-496 within pharmaceutical formulations., Competing Interests: The authors have declared that no competing interests exist.

The nuclear factor κB (NF-κB) pathway plays a key role in innate and adaptive immunity, cell proliferation and survival, inflammation and tumors development. MiR-146a is an immune system regulator that has anti-inflammatory function in multiple cell types and conditions. Here we demonstrate activation of canonical NF-κB pathway in monocytic cells upon HSV-1 replication. By constructing and using a recombinant HSV-1\EGFP virus, we monitored the capability of the virus to recruit NF-κB and we report that the phosphorylation of p65 protein correlates with an active virus replication at single-cell level. In addition, we found that upregulation of miR-146a during viral replication is strictly dependent on NF-κB activation and correlates with tight control of the interleukin-1 receptor-associate kinase 1 (IRAK1). Accordingly, THP-1 DN IκBα cells, expressing a dominant negative mIκBα, did not show upregulation of miR-146a upon HSV-1 infection. Our data suggest that the expression of miRNA-146a modulates NF-κB activation through targeting IRAK1 during HSV-1 replication in THP-1 cells.

Two new tetralkylammonium-OPEs, bearing one or two positively charged groups directly linked to the aromatic residues and two β-d-glucopyranose terminations, were synthesized. Their peculiar structural features, joining the biologically relevant sugar moieties, flat aromatic cores and positive charges, make these luminescent dyes soluble in aqueous media and able to strongly interact with DNA. As a result of UV/Vis spectral variations, DNA melting temperature measures, viscometric titrations and induced CD, we propose a partial insertion of the OPEs aromatic core into the helix, stabilized by glucose H-bonding with the groups accessible from the grooves. This interaction leads to the quenching of the OPE luminescence due to guanine reduction. The biocompatibility of the monocationic OPE with healthy and cancer cells, and the reduction of proliferation in HEp-2 cancer cells induced by the dicationic one, make this class of compounds promising for future biological applications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The paper reviews the network of cellular signaling pathways activated by Functional Graphene Nanomaterials (FGN) designed as a platform for multi-targeted therapy or scaffold in tissue engineering. Cells communicate with each other through a molecular device called signalosome. It is a transient co-cluster of signal transducers and transmembrane receptors activated following the binding of transmembrane receptors to extracellular signals. Signalosomes are thus efficient and sensitive signal-responding devices that amplify incoming signals and convert them into robust responses that can be relayed from the plasma membrane to the nucleus or other target sites within the cell. The review describes the state-of-the-art biomedical applications of FGN focusing the attention on the cell/FGN interactions and signalosome activation.

Functionalized carbon nanotubes (CNTs) have been proposed in the last years as vectors for delivery of biomolecules, proteins and DNA into various cells. In this study, a new multiwalled carbon nanotube β-cyclodextrin platform (MWCNT-CD) modified with branched polyethylenimine (PEI) and doped with Rhodamine (Rhod), MWCNT-CD-PEI-Rhod, was designed and investigated as drug delivery system. The drug binding abilities of MWCNT-CD-PEI-Rhod towards Cidofovir (Cid) and DNA plasmid encoding enhanced green fluorescence protein (pCMS-EGFP) were investigated by complementary spectroscopic techniques. MWCNT-CD-PEI-Rhod showed no significative cytotoxicity and an excellent ability to entrap and delivery Cid. The present study broadens the spectrum of biological evaluation by investigating platform-treatment induced cellular response such as antiviral activity, transfection properties, cellular uptake, internalization mechanisms and cellular localization. The mechanism of cellular uptake was elucidated monitoring the dependence of intracellular red fluorescence from the assembly concentration, time and presence of specific uptake inhibitors. The biological results indicated that MWCNT-CD-PEI-Rhod loaded with Cid and/or pCMS-EGFP crossed the cell membrane via clathrin-dependent pathway and co-localized in lysosomal compartment. However, no green fluorescent protein expression of pCMS-EGFP was detected, whereas the efficient escape of Cid from lysosome and the release close to nuclear region prompted the antiviral activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B β-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that β-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that β-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV., Competing Interests: The authors declare no conflict of interest.

The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.

The aim of the present research was to determine the effect of almond skin extracts on herpes simplex virus 1 (HSV-1) replication. Drug-resistant strains of HSV frequently develop following therapeutic treatment. Therefore, the discovery of novel anti-HSV drugs deserves great effort. Here, we tested both natural (NS) and blanched (BS) polyphenols-rich almond skin extracts against HSV-1. HPLC analysis showed that the prevalent compounds in NS and BS extracts contributing to their antioxidant activity were quercetin, epicatechin and catechin. Results of cell viability indicated that NS and BS extracts were not toxic to cultured Vero cells. Furthermore, NS extracts were more potent inhibitors of HSV-1 than BS extracts, and this trend was in agreement with different concentrations of flavonoids. The plaque forming assay, Western blot and real-time PCR were used to demonstrate that NS extracts were able to block the production of infectious HSV-1 particles. In addition, the viral binding assay demonstrated that NS extracts inhibited HSV-1 adsorption to Vero cells. Our conclusion is that natural products from almond skin extracts are an extraordinary source of antiviral agents and provide a novel treatment against HSV-1 infections.

In the development of new antibacterial therapeutic approaches to fight multidrug-resistant bacteria, antimicrobial photodynamic therapy (aPDT) represents a well-known alternative to treat local infections caused by different microorganisms. Here we present a polypropylene (PP) fabric finished with citrate-hydroxypropyl-βCD polymer (PP-CD) entrapping the tetra-anionic 5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphine (TPPS) as photosensitizer-eluting scaffold (PP-CD/TPPS) for aPDT. The concept is based on host-guest complexation of porphyrin in the cavities of CDs immobilized on the PP fibers, followed by its sustained and controlled delivery in release medium and simultaneous photoinactivation of microorganisms. Morphology of fabric was characterized by optical (OM) and scanning electron microscopies (SEM). Optical properties were investigated by UV-vis absorption, steady- and time-resolved fluorescence emission spectroscopy. X-ray photoelectron spectroscopy (XPS) and FT-IR revealed the surface chemical composition and the distribution map of the molecular components on the fabric, respectively. Direct 1 O 2 determination allowed to assess the potential photodynamic activity of the fabric. Release kinetics of TPPS in physiological conditions pointed out the role of the CD cavity to control the TPPS elution. Photoantimicrobial activity of the porphyrin-loaded textile was investigated against both Gram-positive Staphylococcus aureus ATCC 29213 (S. aureus) and Gram-negative Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa). Optical microscopy coupled with UV-vis extinction and fluorescence spectra aim to ascertain the uptake of TPPS to S. aureus bacterial cells. Finally, PP-CD/TPPS fabric-treated S. aureus cells were photokilled of 99.98%. Moreover, low adhesion of S. aureus cells on textile was established. Conversely, no photodamage of fabric-treated P. aeruginosa cells was observed, together with their satisfying adhesion.

CCR5 stimulation with natural ligands, such as RANTES, classically induces short-term internalization with transient activation of β-arrestins and rapidly recycling on the cell surface. Here we discovered that, in T cells, natural CCR5 antibodies induce a CCR5-negative phenotype with the involvement of β-arrestin2, which leads to the formation of a stable CCR5 signalosome with both β-arrestin2 and ERK1. The activation of β-arrestin2 is necessary to CCR5 signaling for the signalosome formation and stabilization. When all stimuli were washed out, β-arrestin1 silencing favors the activity of β-arrestin2 for the CCR5 signalosome retention. Interestingly, CCR5 turn from Class A trafficking pattern, normally used for its internalization with natural modulating molecules (i.e. RANTES), into a long lasting Class B type specifically induced by stimulation with natural anti-CCR5 antibodies. This new CCR5 pathway is relevant not only to study in depth the molecular basis of all pathologies where CCR5 is involved but also to generate new antidody-based therapeutics.

The combination of conventional anticancer therapy with other treatment modalities such as photodynamic therapy (PDT) is paving the way to novel more effective treatment of solid tumors via light exposure. With this idea in mind, in this paper, nanoparticles (NPs) based on Heptakis (2-oligo(ethyleneoxide)-6-hexadecylthio-)-β-CD (SC16OH) for dual delivery of Zinc-Phthalocyanine (ZnPc) and Docetaxel (DTX) were developed pointing to their potential application as nanomedicine for the combined photodynamic and chemo-therapy of solid tumors. NPs prepared by the emulsion-solvent evaporation technique displayed a hydrodynamic diameter of ≅ 200nm, a negative zeta potential (≅ -27mV) and a satisfactory entrapment efficiency of both drugs at a specific mass ratio. On these bases, NPs containing DTX and ZnPc with theoretical loading of 5% and 0.2% respectively (ZnPc/DTX5-NPs) were selected for further investigations. The allocation of ZnPc and DTX into the colloid was investigated by complementary spectroscopic techniques. In particular, fluorescence emission studies showed the entrapment of ZnPc as a monomer in the carrier, with a low tendency to self-aggregate and consequently a fairly high propensity to photogenerate singlet oxygen. The interaction of SC16OH with DTX, co-entrapped with ZnPc, was elucidated by (1)H NMR and 2D ROESY, which suggested the presence of the chemotherapeutic in the hydrophobic portion of SC16OH. ZnPc/DTX5-NPs were fairly stable in different biological relevant media within 24h. Finally, in vitro potential of the nanoassembly was evaluated in HeLa cancer cells by cell viability exploring both effects of DTX and ZnPc. Overall, results suggest the suitability of NPs based on SC16OH for delivering a combination of DTX with ZnPc to cancer cells, thus inducing photodynamic and antimitotic effects., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.

Photodynamic therapy (PDT) is a minimally invasive procedure that can provide a selective eradication of neoplastic diseases by the combined effect of a photosensitizer, light and oxygen. New amino oligo(phenylene-ethynylene)s (OPEs), bearing hydrophilic glucoside terminations, have been prepared, characterized and tested as photosensitizers in PDT. The effectiveness of these compounds in combination with UVA light has been checked on two tumor cell lines (HEp-2 and HeLa cells, derived from a larynx carcinoma and a cervical carcinoma, respectively). The compounds triggered a mitotic blockage that led to the cell death, being the effect active up to 3 μm concentration. The photophysical properties of OPEs, such as high quantum yield, stability, singlet oxygen production, biocompatibility, easy cell-internalization and very good response even at low concentration, make them promising photosensitizers in the application of PDT., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-lasting internalization (48 h) of the protein, whereas all known CCR5 modulating molecules show a short-term kinetics (60-90 min). Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto unrecognized mechanism of CCR5 modulation mediated by G protein-dependent ERK1 activity. We further demonstrate that ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such as the use of recombinant Abs., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The design of β-cyclodextrin/multiwalled carbon nanotubes hybrid (β-CD-MWCNT) as nanoplatform for the entrapment and delivery of guanine based drugs is described here. The functionalized carbon nanomaterials have been characterized by XPS spectroscopy, electron microscopy (FEG-SEM and TEM), AFM, TGA, and FT-IR to achieve insights on structure, morphology and chemical composition. The drug binding abilities of nanocarrier towards the guanine (G) and Acyclovir (Acy) were proved by UV-vis and DSC experiments. Host-guest equilibrium association constants and drug loading have been evaluated for G/β-CD-MWCNT and Acy/β-CD-MWCNT complexes. The release studies showed a sustained delivery of Acy without initial burst effect confirming a strong interaction of drug with the nanoplatform sites. The preliminary antiviral data indicated that the Acyclovir loaded into the β-CD-MWCNT platform interferes with HSV-1 replication and the antireplicative effect was higher than the free drug., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new family of oligo(phenylene ethynylene) (OPE) glucosides has been prepared and characterized. Our results demonstrate that fine-tuning of their photophysical properties can be obtained by acting on the electronics of the core and molecular skeleton. Modulation of the hydrophobic chain length and substituents on the central moieties influences the bioaffinity too. In particular, introducing a NMe2 group on the aromatic central core affords a highly efficient biocompatible fluorescent probe that can be taken up in cytoplasmic vesicles of HEp-2 cells (cells from epidermoid carcinoma larynx tissue). The photophysical behavior, high quantum yield, and stability open the way to the use of the OPE family as stains for cellular imaging analysis by fluorescence microscopy.

A straightforward transformation of indole-3,4-diones is reported. The reaction feasibility is evidenced by kinetic studies on a model substrate, revealing a double phase process with a first faster pseudo-first-order step (i.e., deprotonation of the dione and self-nucleophilic attack of the anion) and a subsequent slower dehydration of the intermediate. The overall process is faster at pH higher than the pK value of the investigated substrate. The biological relevance of new compounds has been assessed in vitro against herpes simplex virus type-1 (HSV-1), showing a more promising biological profile with respect to their precursors.

Earlier studies have shown that active MEK blocks the activation of protein kinase R (PKR), a component of antiviral innate immune responses. In this report we show that the herpes simplex virus 1 virion host shutoff (VHS) RNase protein and MEK (mitogen-activated protein kinase kinase) act cooperatively in blocking the activation of PKR. This conclusion is based on the following. (i) In contrast to viral gene expression in the parental cell line or a cell line expressing a constitutively active MEK, the replication of a VHS mutant is particularly impaired in cells expressing dominant negative MEK. In this cell line PKR is activated by phosphorylation, and the accumulation of several viral proteins is delayed. (ii) In transfected cells, wild-type VHS blocked the activation of PKR, whereas PKR was activated in cells transfected with a mutant VHS or with plasmids encoding the VHS RNase and VP16 and VP22, the two viral proteins that neutralize the RNase activity of VHS. The results suggest that early in infection the VHS RNase degrades RNAs that activate PKR. Coupled with published data, the results suggest that inhibition of activation of PKR or its effect on viral replication is staged early in infection by VHS, postsynthesis of VP16 and VP22 by the γ(1)34.5 protein, and very late in infection by the U(S)11 protein.

Herein we report the design, preparation, and properties of a supramolecular system based on a tailored nitric oxide (NO) photodonor and a rhodamine-labeled β-cyclodextrin conjugate. The combination of spectroscopic and photochemical experiments shows the absence of significant interchromophoric interactions between the host and the guest in the excited states. As a result, the complex is able to release NO under the exclusive control of visible light, as unambiguously demonstrated by direct detection of this transient species through an amperometric technique, and exhibits the typical red fluorescence of the rhodamine appendage. The supramolecular complex effectively internalizes in HeLa cancer cells as proven by fluorescence microscopy, shows a satisfactory biocompatibility in the dark, and induces about 50% of cell mortality upon irradiation with visible light. The convergence of all these properties in one single complex makes the present host-guest ensemble an appealing candidate for further delevopment of photoactivatable nanoscaled systems addressed to photostimulated NO-based therapy., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enolizable 6-membered cyclic 1,3-dicarbonyls undergo an efficient and diastereoselective domino condensation/addition/heterocyclization reaction with arylaldehydes and phenacyl chloride, producing highly substituted dihydrofuran-fused derivatives. Ring size of the cyclic 1,3-dicarbonyls and the presence of at least one keto group are crucial to the reaction's success. The new compounds were evaluated in vitro for antiviral activity against herpes simplex virus type-1 (HSV-1). Interestingly, some of them appeared able to interfere with HSV-1 replication, without detection of cytotoxic effects.

We have developed a supramolecular nanoassembly capable of inducing remarkable levels of cancer cell mortality through a bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen ((1)O(2)). This was achieved through the appropriate incorporation of an anionic porphyrin (as (1)O(2) photosensitizer) and of a tailored NO photodonor in different compartments of biocompatible nanoparticles based on cationic amphiphilic cyclodextrins. The combination of steady-state and time-resolved spectroscopic techniques showed the absence of significant intra- and interchromophoric interaction between the two photoactive centers embedded in the nanoparticles, with consequent preservation of their photodynamic properties. Photodelivery of NO and (1)O(2) from the nanoassembly on visible light excitation was unambiguously demonstrated by direct and real-time monitoring of these transient species through amperometric and time-resolved infrared luminescence measurements, respectively. The typical red fluorescence of the porphyrin units was essentially unaffected in the bichromophoric nanoassembly, allowing its localization in living cells. The convergence of the dual therapeutic action and the imaging capacities in one single structure makes this supramolecular architecture an appealing, multifunctional candidate for applications in biomedical research., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)