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Enrico Gallo,1 Carlo Diaferia,2 Elisabetta Rosa,2 Giovanni Smaldone,1 Giancarlo Morelli,2 Antonella Accardo2 1IRCCS SDN, Naples, 80143, Italy; 2Department of Pharmacy and Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, Naples, 80134, ItalyCorrespondence: Antonella AccardoDepartment of Pharmacy and Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, Via Mezzocannone 16, Naples, 80134, ItalyTel +39 0812532045Fax +39 0812536642Email antonella.accardo@unina.itIntroduction: The clinical use of the antitumoral drug doxorubicin (Dox) is reduced by its dose-limiting toxicity, related to cardiotoxic side effects and myelosuppression. In order to overcome these drawbacks, here we describe the synthesis, the structural characterization and the in vitro cytotoxicity assays of hydrogels (HGs) and nanogels (NGs) based on short peptide sequences loaded with Dox or with its liposomal formulation, Doxil.Methods: Fmoc-FF alone or in combination with (FY)3 or PEG8-(FY)3 peptides, at two different ratios (1/1 and 2/1 v/v), were used for HGs and NGs formulations. HGs were prepared according to the “solvent-switch” method, whereas NGs were obtained through HG submicronition by the top-down methodology in presence of TWEEN® 60 and SPAN® 60 as stabilizing agents. HGs gelation kinetics were assessed by Circular Dichroism (CD). Stability and size of NGs were studied using Dynamic Light Scattering (DLS) measurements. Cell viability of empty and filled Dox HGs and NGs was evaluated on MDA-MB-231 breast cancer cells. Moreover, cell internalization of the drug was evaluated using immunofluorescence assays.Results: Dox filled hydrogels exhibit a high drug loading content (DLC=0.440), without syneresis after 10 days. Gelation kinetics (20– 40 min) and the drug release (16– 28%) over time of HGs were found dependent on relative peptide c

Enrico Gallo,1 Carlo Diaferia,2 Elisabetta Rosa,2 Giovanni Smaldone,1 Giancarlo Morelli,2 Antonella Accardo2 1IRCCS SDN, Naples, 80143, Italy; 2Department of Pharmacy and Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, Naples, 80134, ItalyCorrespondence: Antonella AccardoDepartment of Pharmacy and Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, Via Mezzocannone 16, Naples, 80134, ItalyTel +39 0812532045Fax +39 0812536642Email antonella.accardo@unina.itIntroduction: The clinical use of the antitumoral drug doxorubicin (Dox) is reduced by its dose-limiting toxicity, related to cardiotoxic side effects and myelosuppression. In order to overcome these drawbacks, here we describe the synthesis, the structural characterization and the in vitro cytotoxicity assays of hydrogels (HGs) and nanogels (NGs) based on short peptide sequences loaded with Dox or with its liposomal formulation, Doxil.Methods: Fmoc-FF alone or in combination with (FY)3 or PEG8-(FY)3 peptides, at two different ratios (1/1 and 2/1 v/v), were used for HGs and NGs formulations. HGs were prepared according to the “solvent-switch” method, whereas NGs were obtained through HG submicronition by the top-down methodology in presence of TWEEN® 60 and SPAN® 60 as stabilizing agents. HGs gelation kinetics were assessed by Circular Dichroism (CD). Stability and size of NGs were studied using Dynamic Light Scattering (DLS) measurements. Cell viability of empty and filled Dox HGs and NGs was evaluated on MDA-MB-231 breast cancer cells. Moreover, cell internalization of the drug was evaluated using immunofluorescence assays.Results: Dox filled hydrogels exhibit a high drug loading content (DLC=0.440), without syneresis after 10 days. Gelation kinetics (20– 40 min) and the drug release (16– 28%) over time of HGs were found dependent on relative peptide c

Paola Ringhieri,1 Silvia Mannucci,2 Giamaica Conti,2 Elena Nicolato,2 Giulio Fracasso,3 Pasquina Marzola,4 Giancarlo Morelli,1 Antonella Accardo1 1Department of Pharmacy and Interuniversity Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, Napoli, 2Department of Neurological Biomedical and Movement Sciences, 3Section of Immunology, Department of Medicine, 4Department of Informatics, University of Verona, Verona, Italy Abstract: Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents. Keywords: anti-HER2 liposomes, target peptide, KCCYSL peptide, breast cancer, click chemistry, branched peptides&nbsp

Paola Ringhieri,1 Silvia Mannucci,2 Giamaica Conti,2 Elena Nicolato,2 Giulio Fracasso,3 Pasquina Marzola,4 Giancarlo Morelli,1 Antonella Accardo1 1Department of Pharmacy and Interuniversity Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, Napoli, 2Department of Neurological Biomedical and Movement Sciences, 3Section of Immunology, Department of Medicine, 4Department of Informatics, University of Verona, Verona, Italy Abstract: Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents. Keywords: anti-HER2 liposomes, target peptide, KCCYSL peptide, breast cancer, click chemistry, branched peptides&nbsp

Antonella Accardo,1 Mariateresa Vitiello,2,3 Diego Tesauro,1 Marilena Galdiero,2 Emiliana Finamore,2 Francesca Martora,2 Rosalba Mansi,1 Paola Ringhieri,1 Giancarlo Morelli11Department of Pharmacy, Interuniversitary Centre for Research on Bioactive peptides, CIRPeB, University of Naples "Federico II", Institute of Biostructures and Bioimaging IBB-CNR, Naples, Italy; 2Department of Experimental Medicine, Section of Microbiology and Clinical Microbiology, Second University of Naples, Naples, Italy; 3Department of Clinical Pathology and Transfusion Medicine, University Hospital “Ruggi d'Aragona”, Salerno, ItalyAbstract: The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409-505 and gD301-309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods. Structural characterization of the aggregates confirmed that they were sufficiently stable and compatible with in vivo use: critical micelle concentration values around 4.0 · 10-7 mol · Kg-1; hydrodynamic radii (RH) between 50–80 nm, and a zeta potential (ζ) around – 40 mV were found for all aggregates. The in vitro results indicate that both peptide epitopes and micelles, at 10 µM, triggered U937 and RAW 264.7 cells to release appreciable levels of cytokines. In particular, interleukin (IL)-23-, IL-6-, IL-8- or macrophage inflammatory protein (MIP)-2-, and tumor necrosis factor (TNF)-α-release increased considerably when cells were treated with the gB-micelles or gD-micelles compared

Antonella Accardo,1 Luigi Aloj,2 Michela Aurilio,2 Giancarlo Morelli,1 Diego Tesauro11Centro interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), Department of Pharmacy and Istituto di Biostrutture e Bioimmagini - Consiglio Nazionale delle Ricerche (IBB CNR), University of Naples “Federico II”, 2Department of Nuclear Medicine, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”, Napoli, ItalyAbstract: Active targeting by means of drug encapsulated nanoparticles decorated with targeting bioactive moieties represents the next frontier in drug delivery; it reduces drug side effects and increases the therapeutic index. Peptides, based on their chemical and biological properties, could have a prevalent role to direct drug encapsulated nanoparticles, such as liposomes, micelles, or hard nanoparticles, toward the tumor tissues. A considerable number of molecular targets for peptides are either exclusively expressed or overexpressed on both cancer vasculature and cancer cells. They can be classified into three wide categories: integrins; growth factor receptors (GFRs); and G-protein coupled receptors (GPCRs). Therapeutic agents based on nanovectors decorated with peptides targeting membrane receptors belonging to the GPCR family overexpressed by cancer cells are reviewed in this article. The most studied targeting membrane receptors are considered: somatostatin receptors; cholecystokinin receptors; receptors associated with the Bombesin like peptides family; luteinizing hormone-releasing hormone receptors; and neurotensin receptors. Nanovectors of different sizes and shapes (micelles, liposomes, or hard nanoparticles) loaded with doxorubicin or other cytotoxic drugs and externally functionalized with natural or synthetic peptides are able to target the overexpressed receptors and are described based on their formulation and in vitro and in vivo behaviors.Keywords: receptors binding peptides, drug de

Antonella Accardo,1 Mariateresa Vitiello,2,3 Diego Tesauro,1 Marilena Galdiero,2 Emiliana Finamore,2 Francesca Martora,2 Rosalba Mansi,1 Paola Ringhieri,1 Giancarlo Morelli11Department of Pharmacy, Interuniversitary Centre for Research on Bioactive peptides, CIRPeB, University of Naples "Federico II", Institute of Biostructures and Bioimaging IBB-CNR, Naples, Italy; 2Department of Experimental Medicine, Section of Microbiology and Clinical Microbiology, Second University of Naples, Naples, Italy; 3Department of Clinical Pathology and Transfusion Medicine, University Hospital “Ruggi d'Aragona”, Salerno, ItalyAbstract: The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409-505 and gD301-309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods. Structural characterization of the aggregates confirmed that they were sufficiently stable and compatible with in vivo use: critical micelle concentration values around 4.0 · 10-7 mol · Kg-1; hydrodynamic radii (RH) between 50–80 nm, and a zeta potential (ζ) around – 40 mV were found for all aggregates. The in vitro results indicate that both peptide epitopes and micelles, at 10 µM, triggered U937 and RAW 264.7 cells to release appreciable levels of cytokines. In particular, interleukin (IL)-23-, IL-6-, IL-8- or macrophage inflammatory protein (MIP)-2-, and tumor necrosis factor (TNF)-α-release increased considerably when cells were treated with the gB-micelles or gD-micelles compared

Antonella Accardo,1 Luigi Aloj,2 Michela Aurilio,2 Giancarlo Morelli,1 Diego Tesauro11Centro interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), Department of Pharmacy and Istituto di Biostrutture e Bioimmagini - Consiglio Nazionale delle Ricerche (IBB CNR), University of Naples “Federico II”, 2Department of Nuclear Medicine, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”, Napoli, ItalyAbstract: Active targeting by means of drug encapsulated nanoparticles decorated with targeting bioactive moieties represents the next frontier in drug delivery; it reduces drug side effects and increases the therapeutic index. Peptides, based on their chemical and biological properties, could have a prevalent role to direct drug encapsulated nanoparticles, such as liposomes, micelles, or hard nanoparticles, toward the tumor tissues. A considerable number of molecular targets for peptides are either exclusively expressed or overexpressed on both cancer vasculature and cancer cells. They can be classified into three wide categories: integrins; growth factor receptors (GFRs); and G-protein coupled receptors (GPCRs). Therapeutic agents based on nanovectors decorated with peptides targeting membrane receptors belonging to the GPCR family overexpressed by cancer cells are reviewed in this article. The most studied targeting membrane receptors are considered: somatostatin receptors; cholecystokinin receptors; receptors associated with the Bombesin like peptides family; luteinizing hormone-releasing hormone receptors; and neurotensin receptors. Nanovectors of different sizes and shapes (micelles, liposomes, or hard nanoparticles) loaded with doxorubicin or other cytotoxic drugs and externally functionalized with natural or synthetic peptides are able to target the overexpressed receptors and are described based on their formulation and in vitro and in vivo behaviors.Keywords: receptors binding peptides, drug de

Antonella Accardo1,2*, Giuseppina Salsano3*, Anna Morisco4, Michela Aurilio4, Antonio Parisi5, Francesco Maione5, Carla Cicala5, Diego Tesauro1,2, Luigi Aloj4, Giuseppe De Rosa3, Giancarlo Morelli1,21CIRPeB, Department of Biological Sciences and IBB CNR, University of Naples “Federico II”, 2Invectors srl, 3Department of Pharmaceutical Chemistry, University of Naples “Federico II”, 4Department of Nuclear Medicine, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”, 5Department of Experimental Pharmacology, University of Naples “Federico II”, Napoli, Italy*These authors contributed equally to this workObjectives: Drug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors.Methods: A new amphiphilic peptide derivative (MonY-BN) containing the BN(7–14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C18 alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized.Results: Liposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with pepti

Antonella Accardo1,2*, Giuseppina Salsano3*, Anna Morisco4, Michela Aurilio4, Antonio Parisi5, Francesco Maione5, Carla Cicala5, Diego Tesauro1,2, Luigi Aloj4, Giuseppe De Rosa3, Giancarlo Morelli1,21CIRPeB, Department of Biological Sciences and IBB CNR, University of Naples “Federico II”, 2Invectors srl, 3Department of Pharmaceutical Chemistry, University of Naples “Federico II”, 4Department of Nuclear Medicine, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”, 5Department of Experimental Pharmacology, University of Naples “Federico II”, Napoli, Italy*These authors contributed equally to this workObjectives: Drug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors.Methods: A new amphiphilic peptide derivative (MonY-BN) containing the BN(7–14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C18 alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized.Results: Liposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with pepti