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Rosa Bellavita,1,* Marialuisa Piccolo,1,* Linda Leone,2 Maria Grazia Ferraro,1,3 Principia Dardano,4 Luca De Stefano,4 Flavia Nastri,2 Carlo Irace,1 Annarita Falanga,5 Stefania Galdiero1,* 1Department of Pharmacy, School of Medicine, University of Naples ‘Federico II’, Napoli, Italy; 2Department of Chemical Sciences, University of Napoli “Federico II”, Naples, Italy; 3School of Infection and Immunity, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK; 4Institute of Applied Sciences and Intelligent Systems, Consiglio Nazionale delle Ricerche, Naples, Italy; 5Department of Agricultural Science, University of Naples “Federico II”, Portici, Italy*These authors contributed equally to this workCorrespondence: Stefania Galdiero, Email stefania.galdiero@unina.itIntroduction: The design of delivery tools that efficiently transport drugs into cells remains a major challenge in drug development for most pathological conditions. Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with poor prognosis and limited effective therapeutic options.Purpose: In TNBC treatment, chemotherapy remains the milestone, and doxorubicin (Dox) represents the first-line systemic treatment; however, its non-selective distribution causes a cascade of side effects. To address these problems, we developed a delivery platform based on the self-assembly of amphiphilic peptides carrying several moieties on their surfaces, aimed at targeting, enhancing penetration, and therapy.Methods: Through a single-step self-assembly process, we used amphiphilic peptides to obtain nanofibers decorated on their surfaces with the selected moieties. The surface of the nanofiber was decorated with a cell-penetrating peptide (gH625), an EGFR-targeting peptide (P22), and Dox bound to the cleavage sequence selectively recognized and cleaved by MMP-9 to obtain on-demand drug release. Detailed physicochemical and cellular analyses were performed.Results: The obtained nanofiber (NF-Dox) had a length of 250 nm and a diameter of 10 nm, and it was stable under dilution, ionic strength, and different pH environments. The biological results showed that the presence of gH625 favored the complete internalization of NF-Dox after 1h in MDA-MB 231 cells, mainly through a translocation mechanism. Interestingly, we observed the absence of toxicity of the carrier (NF) on both healthy cells such as HaCaT and TNBC cancer lines, while a similar antiproliferative effect was observed on TNBC cells after the treatment with the free-Dox at 50 μM and NF-Dox carrying 7.5 μM of Dox.Discussion: We envision that this platform is extremely versatile and can be used to efficiently carry and deliver diverse moieties. The knowledge acquired from this study will provide important guidelines for applications in basic research and biomedicine. Keywords: self-assembling peptides, triple negative breast cancer, nanofiber, on-demand strategy, doxorubicin

Annarita Falanga,1 Antonietta Siciliano,2 Mariateresa Vitiello,3 Gianluigi Franci,4 Valentina Del Genio,5 Stefania Galdiero,5 Marco Guida,2 Federica Carraturo,2 Amir Fahmi,6,* Emilia Galdiero2,* 1Department of Agricultural Science, University of Naples Federico II, Portici 80055, Italy; 2Department of Biology, University of Naples Federico II, Naples 80100, Italy; 3Department of Clinical Pathology, Virology Unit, “San Giovanni di Dio e Ruggi d’Aragona Hospital”, Salerno, Italy; 4Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana,” University of Salerno, Baronissi, Italy; 5Department of Pharmacy, School of Medicine, University of Naples Federico II, Naples 80134, Italy; 6Rhein-Waal University of Applied Sciences, Kleve D-47533, Germany*These authors contributed equally to this workCorrespondence: Emilia Galdiero; Amir Fahmi Tel +39081679182Email egaldier@unina.it; Amir.fahmi@hochschule-Rhein-Waal.deBackground: Metallic nanoparticles (NPs) are highly exploited in manufacturing and medical processes in a broad spectrum of industrial applications and in the academic sectors. Several studies have suggested that many metallic nanomaterials including those derived by silver (Ag) are entering the ecosystem to cause significant toxic consequences in cell culture and animal models. However, ecotoxicity studies are still receiving limited attention when designing functionalized and non.-functionalized AgNPs.Objective: This study aimed to investigate different ecotoxicological profiles of AgNPs, which were analyzed in two different states: in pristine form uncoated AgNPs and coated AgNPs with the antimicrobial peptide indolicidin. These two types of AgNPs are exploited for a set of different tests using Daphnia magna and Raphidocelis subcapitata, which are representatives of two different levels of the aquatic trophic chain, and seeds of Lepidium sativum, Cucumis sativus and Lactuca sativa.Results: Ecotoxicological studies showed that the most sensitive organism to AgNPs was crustacean D. magna, followed by R. subcapitata and plant seeds, while AgNPs coated with indolicidin (IndAgNPs) showed a dose-dependent decreased toxicity for all three.Conclusion: The obtained results demonstrate that high ecotoxicity induced by AgNPs is strongly dependent on the surface chemistry, thus the presence of the antimicrobial peptide. This finding opens new avenues to design and fabricate the next generation of metallic nanoparticles to ensure the biosafety and risk of using engineered nanoparticles in consumer products.Keywords: silver nanoparticles, antimicrobial peptide indolicidin, in vivo toxicity, phytotoxicity, nanotoxicology

Elisabetta de Alteriis,1 Valeria Maselli,1 Annarita Falanga,2 Stefania Galdiero,2 Federica Maria Di Lella,3 Renato Gesuele,1 Marco Guida,1 Emilia Galdiero1 1Department of Biology, University of Naples “Federico II”, Naples, Italy; 2Department of Pharmacy, University of Naples “Federico II”, Naples, Italy; 3Section of Microbiology and Virology, University Hospital “Luigi Vanvitelli” of Naples, Naples, Italy Background: This article examines the use of a novel nano-system, gold nanoparticles coated with indolicidin (AuNPs–indolicidin), against pathogenic Candida albicans biofilms. Candida species cause frequent infections owing to their ability to form biofilms, primarily on implant devices. Materials and methods: We used an integrated approach, evaluating the effect of AuNPs-indolicidin on prevention and eradication of Candida biofilms formed in multi-well polystyrene plates, with relative gene expression assays. Four biofilm-associated genes (FG1, HWP1, ALS1 and ALS3, and CDR1 and CDR2) involved in efflux pump were analyzed using reverse transcription polymerase chain reaction. Results: Treatment with the nano-complex significantly inhibits the capacity of C. albicans to form biofilms and impairs preformed mature biofilms. Treatment with AuNPs–indolicidin results in an increase in the kinetics of Rhodamine 6G efflux and a reduction in the expression of biofilm-related genes. Conclusion: These data provide a chance to develop novel therapies against nosocomially acquired refractory C. albicans biofilms. Keywords: antimicrobial activity, anti-biofilm activity, fungal biofilm, drug resistance, fungal infections

Emilia Galdiero,1 Annarita Falanga,2 Antonietta Siciliano,1 Valeria Maselli,1 Marco Guida,1 Rosa Carotenuto,1 Margherita Tussellino,1 Lucia Lombardi,3 Giovanna Benvenuto,4 Stefania Galdiero2 1Department of Biology, 2Department of Pharmacy and CiRPEB, University of Naples Federico II, 3Department of Experimental Medicine, Second University of Naples, 4Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy Abstract: The use of quantum dots (QDs) for nanomedicine is hampered by their potential toxicologic effects and difficulties with delivery into the cell interior. We accomplished an in vivo study exploiting Daphnia magna and Xenopus laevis to evaluate both toxicity and uptake of QDs coated with the membranotropic peptide gH625 derived from the glycoprotein H of herpes simplex virus and widely used for drug delivery studies. We evaluated and compared the effects of QDs and gH625-QDs on the survival, uptake, induction of several responsive pathways and genotoxicity in D. magna, and we found that QDs coating plays a key role. Moreover, studies on X. laevis embryos allowed to better understand their cell/tissue localization and delivery efficacy. X. laevis embryos raised in Frog Embryo Teratogenesis Assay-Xenopus containing QDs or gH625-QDs showed that both nanoparticles localized in the gills, lung and intestine, but they showed different distributions, indicating that the uptake of gH625-QDs was enhanced; the functionalized QDs had a significantly lower toxic effect on embryos’ survival and phenotypes. We observed that D. magna and X. laevis are useful in vivo models for toxicity and drug delivery studies. Keywords: membranotropic peptide, delivery, blood–brain barrier, nanoparticles, genotoxicity

Emilia Galdiero,1 Antonietta Siciliano,1 Valeria Maselli,1 Renato Gesuele,1 Marco Guida,1 Domenico Fulgione,1 Stefania Galdiero,2 Lucia Lombardi,3 Annarita Falanga2 1Department of Biology, University of Naples “Federico II”, Naples, Italy; 2Department of Pharmacy and Cirpeb, University of Naples “Federico II”, Naples, Italy; 3Department of Experimental Medicine, Second University of Naples, Naples, Italy Abstract: This study attempts to evaluate the antimicrobial activity and the ecotoxicity of quantum dots (QDs) alone and coated with indolicidin. To meet this objective, we tested the level of antimicrobial activity on Gram-positive and Gram-negative bacteria, and we designed an ecotoxicological battery of test systems and indicators able to detect different effects using a variety of end points. The antibacterial activity was analyzed against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 1025), Escherichia coli (ATCC 11229), and Klebsiella pneumoniae (ATCC 10031), and the results showed an improved germicidal action of QDs-Ind. Toxicity studies on Daphnia magna indicated a decrease in toxicity for QDs-Ind compared to QDs alone, lack of bioluminescence inhibition on Vibrio fisheri, and no mutations in Salmonella typhimurium TA 100. The comet assay and oxidative stress experiments performed on D. magna showed a genotoxic and an oxidative damage with a dose–response trend. Indolicidin retained its activity when bound to QDs. We observed an enhanced activity for QDs-Ind. The presence of indolicidin on the surface of QDs was able to decrease its QDs toxicity. Keywords: peptide, quantum dots, ecotoxicity, antimicrobial activity, oxidative stress, genotoxicity

Olga Scudiero,1,2 Ersilia Nigro,1 Marco Cantisani,3 Irene Colavita,1 Marilisa Leone,4 Flavia Anna Mercurio,4 Massimiliano Galdiero,5 Antonello Pessi,1 Aurora Daniele,1,6 Francesco Salvatore,1,2,7 Stefania Galdiero3,4 1CEINGE-Biotecnologie Avanzate Scarl, Naples, Italy; 2Dipartimento di Medicina Molecolare e Biotecnologie Mediche, 3Dipartimento di Farmacia, Università di Napoli Federico II, Naples, Italy; 4Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy; 5Dipartimento di Medicina Sperimentale, Seconda Università di Napoli, Naples, Italy; 6Dipartimento di Scienze e Tecnologie Ambientali Biologiche Farmaceutiche, Seconda Università di Napoli, Caserta, Italy; 7IRCCS Fondazione SDN, Naples, Italy Abstract: We have designed a cyclic 17-amino acid β-defensin analog featuring a single disulfide bond. This analog, designated “AMC” (ie, antimicrobial cyclic peptide), combines the internal hydrophobic domain of hBD1 and the C-terminal charged region of hBD3. The novel peptide was synthesized and characterized by nuclear magnetic resonance spectroscopy. The antimicrobial activities against gram-positive and gram-negative bacteria as well as against herpes simplex virus type 1 were analyzed. The cytotoxicity and serum stability were assessed. Nuclear magnetic resonance of AMC in aqueous solution suggests that the structure of the hBD1 region, although not identical, is preserved. Like the parent defensins, AMC is not cytotoxic for CaCo-2 cells. Interestingly, AMC retains the antibacterial activity of the parent hBD1 and hBD3 against Pseudomonas aeruginosa, Enterococcus faecalis, and Escherichia coli, and exerts dose-dependent activity against herpes simplex virus type 1. Moreover, while the antibacterial and antiviral activities of the oxidized and reduced forms of the parent defensins are similar, those of AMC are significantly different, and oxidized AMC is also considerably more stable in human serum. Taken together, our data also suggest that this novel peptide may be added to the arsenal of tools available to combat antibiotic-resistant infectious diseases, particularly because of its potential for encapsulation in a nanomedicine vector. Keywords: antimicrobial activity, cyclic mini-peptide, human beta-defensin

Salvatore Valiante,1,* Annarita Falanga,2,3,* Luisa Cigliano,1 Giuseppina Iachetta,1 Rosa Anna Busiello,1 Valeria La Marca,1 Massimiliano Galdiero,4 Assunta Lombardi,1 Stefania Galdiero1,2 1Department of Biology, 2Department of Pharmacy, 3DFM Scarl, University of Naples Federico II, 4Department of Experimental Medicine, II University of Naples, Naples, Italy *These authors contributed equally to this paper and are considered joint first authors Abstract: Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood–brain barrier (BBB). In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases. Keywords: drug delivery, neurons, astrocytes, blood–brain barrier, peptide

Swapnil Gaikwad,1 Avinash Ingle,1 Aniket Gade,1 Mahendra Rai,1 Annarita Falanga,3 Novella Incoronato,2 Luigi Russo,2 Stefania Galdiero,3 Massimilano Galdiero2 1Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, Maharashtra, India; 2Department of Experimental Medicine, Division of Microbiology, II University of Naples, 3Department of Pharmacy, University of Naples “Federico II”, DFM and Institute of Biostructures and Bioimages, Naples, Italy Abstract: The interaction between silver nanoparticles and viruses is attracting great interest due to the potential antiviral activity of these particles, and is the subject of much research effort in the treatment of infectious diseases. In this work, we demonstrate that silver nanoparticles undergo a size-dependent interaction with herpes simplex virus types 1 and 2 and with human parainfluenza virus type 3. We show that production of silver nanoparticles from different fungi is feasible, and their antiviral activity is dependent on the production system used. Silver nanoparticles are capable of reducing viral infectivity, probably by blocking interaction of the virus with the cell, which might depend on the size and zeta potential of the silver nanoparticles. Smaller-sized nanoparticles were able to inhibit the infectivity of the viruses analyzed. Keywords: silver nanoparticles, antiviral, herpes simplex virus, parainfluenza virus

Giovanni Smaldone,1,2 Annarita Falanga,3 Domenica Capasso,4 Daniela Guarnieri,5,6 Stefania Correale,1,7 Massimiliano Galdiero,8 Paolo A Netti,4 Massimo Zollo,9 Stefania Galdiero,1,2 Sonia Di Gaetano,1 Emilia Pedone1 1Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy; 2Department of Pharmacy and Interuniversity Research Center on Bioactive Peptides, Federico II University of Naples, Naples, Italy; 3Molecular Diagnostics and Pharmaceuticals Scarl, Naples, Italy; 4Special Center for Biotechnology, Federico II University of Naples, Naples, Italy; 5Center for Advanced Biomaterials for Health Care, Interdisciplinary Research Centre on Biomaterials, Italian Institute of Technology, Naples, Italy; 6Interdisciplinary Research Centre on Biomaterials, Federico II University of Naples, Naples, Italy; 7Kedrion S.p.A, Sant'antimo, Naples, Italy; 8Department of Experimental Medicine, Federico II University of Naples, Naples, Italy; 9CEINGE – Advanced Biotechnology Scarl, Naples, Italy Abstract: A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625–644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier. Keywords: delivery, IDP

Rossella Tarallo,1 Tom P Carberry,2 Annarita Falanga,1 Mariateresa Vitiello,3 Stefania Galdiero,1 Massimiliano Galdiero,3 Marcus Weck21Dipartimento di Farmacia, Università di Napoli "Federico II," and DFM Scarl, Napoli, Italia; 2Molecular Design Institute and Department of Chemistry, New York University, New York, NY, USA; 3Dipartimento di Medicina Sperimentale, Seconda Università degli Studi di Napoli, Napoli, ItaliaAbstract: This contribution reports the synthesis of a poly(amide)-based dendrimer functionalized at the termini with a membrane-interacting peptide derived from the herpes simplex virus (HSV) type 1 glycoprotein H, namely gH625-644. This peptide has been shown to interact with model membranes and to inhibit viral infectivity. The peptidodendrimer inhibits both HSV-1 and HSV-2 at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite of viral internalization. The 50% inhibitory concentration was 100 and 300 nM against HSV-1 and HSV-2 respectively, with no evidence of cell toxicity at these concentrations. These results show that the functionalization of a dendrimer with the peptide sequence derived from an HSV glycoprotein shows promising inhibitory activity towards viruses of the Herpesviridae family.Keywords: peptidodendrimer, antiviral activity, membranotropic peptides

Marco Cantisani,1 Mariateresa Vitiello,2 Annarita Falanga,1 Emiliana Finamore,2 Marilena Galdiero,2 Stefania Galdiero11Department of Biological Sciences, CIRPeB and IBB CNR, University of Naples "Federico II," Napoli, Italy; 2Department of Experimental Medicine, II University of Naples, Napoli, ItalyAbstract: Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children. It is characterized by inflammation that is mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. A synthetic peptide corresponding to loop L7 of the porin activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways. We report a novel use of the complementary peptide approach to design a peptide that is able to bind selectively to the protein P2, thereby reducing its activity. This work provides insights into essential molecular details of P2 that may affect the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.Keywords: complementary-peptide, rational design, porin