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Barbara Bortot, 1 Maurizio Mongiat, 2 Erica Valencic, 3 Simeone Dal Monego, 4 Danilo Licastro, 4 Matteo Crosera, 5 Gianpiero Adami, 5 Enrico Rampazzo, 6 Giuseppe Ricci, 7, 8 Federico Romano, 7 Giovanni Maria Severini, 1 Stefania Biffi 7 1Department of Medical Genetics, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy; 2Department of Research and Diagnosis, Division of Molecular Oncology, Centro Di Riferimento Oncologico Di Aviano (CRO) IRCCS, Aviano, Italy; 3Department of Pediatrics, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy; 4ARGO Open Lab Platform for Genome Sequencing, AREA Science Park, Trieste, Italy; 5Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy; 6Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, Italy; 7Department of Obstetrics and Gynecology, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy; 8Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, ItalyCorrespondence: Stefania Biffi Email stefania.biffi@burlo.trieste.itBackground: Platinum resistance is a major challenge in the management of ovarian cancer. Even low levels of acquired resistance at the cellular level lead to impaired response to cisplatin. In ovarian cancer intraperitoneal therapy, nanoparticle formulation can improve the cisplatin’s pharmacokinetics and safety profile.Purpose: This work aimed to investigate the chemo-sensitivity of ovarian cancer SKOV3 cells upon short-term (72h) single treatment of cisplatin and cisplatin-loaded biodegradable nanoparticles (Cis-NP). The aim was then to determine the therapeutic properties of Cis-NP in vivo using a SKOV3-luc cells’ xenograft model in mice.Methods: Cell cytotoxicity was assessed after the exposure of the cell culture to cisplatin or Cis-NP. The effect of treatments on EMT and CSC-like phenotype was studied by analyzing a panel of markers by flow cytometry. Intracellular platinum concentration was determined by inductively coupled plasma mass spectrometry (ICS-MS), and gene expression was evaluated by RNAseq analysis. The efficacy of intraperitoneal chemotherapy was evaluated in a SKOV3-luc cells’ xenograft model in mice, through a combination of bioluminescence imaging, histological, and immunohistochemical analyses.Results: We observed in vitro that short-term treatment of cisplatin has a critical role in determining the potential induction of chemoresistance, and a nanotechnology-based drug delivery system can modulate it. The RNAseq actg 3nalysis underlines a protective effect of nanoparticle system according to their ability to down-regulate several genes involved in chemoresistance, cell proliferation, and apoptosis. The highest intracellular platinum concentration obtained with Cis-NP treatment significantly improved the efficacy. Consistent with in vitro results, we found that Cis-NP treatment in vivo can significantly reduce tumor burden and aggressiveness compared to the free drug.Conclusion: Nanoparticle-mediated cisplatin delivery may serve as an intracellular depot impacting the cisplatin pharmacodynamic performance at cellular levels. These features may contribute to improving the drawbacks of conventional intraperitoneal therapy, and therefore will require further investigations in vivo.Keywords: cisplatin resistance, ovarian cancer, SKOV3, nanoparticle, epithelial-mesenchymal transition, Ca125, apoptosis

Stefania Biffi,1 Luca Petrizza,2 Chiara Garrovo,1 Enrico Rampazzo,2 Laura Andolfi,3 Pierangela Giustetto,4 Ivaylo Nikolov,5 Gabor Kurdi,5 Miltcho Boyanov Danailov,5 Giorgio Zauli,1 Paola Secchiero,6 Luca Prodi2 1Institute for Maternal and Child Health – IRCCS “Burlo Garofolo”, Trieste, 2Department of Chemistry ‘‘G Ciamician’’, University of Bologna, Bologna, 3IOM-CNR TASC Laboratory, Basovizza, Trieste, 4Ephoran – Multi-Imaging Solutions, Bioindustry Park Silvano Fumero, Torino, 5Elettra-Sincrotrone Trieste, Trieste, 6Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy Purpose: The aim of the present study was to develop nanoprobes with theranostic features, including – at the same time – photoacoustic, near-infrared (NIR) optical imaging, and photothermal properties, in a versatile and stable core–shell silica-polyethylene glycol (PEG) nanoparticle architecture. Materials and methods: We synthesized core–shell silica-PEG nanoparticles by a one-pot direct micelles approach. Fluorescence emission and photoacoustic and photothermal properties were obtained at the same time by appropriate doping with triethoxysilane-derivatized cyanine 5.5 (Cy5.5) and cyanine 7 (Cy7) dyes. The performances of these nanoprobes were measured in vitro, using nanoparticle suspensions in phosphate-buffered saline and blood, dedicated phantoms, and after incubation with MDA-MB-231 cells. Results: We obtained core–shell silica-PEG nanoparticles endowed with very high colloidal stability in water and in biological environment, with absorption and fluorescence emission in the NIR field. The presence of Cy5.5 and Cy7 dyes made it possible to reach a more reproducible and higher doping regime, producing fluorescence emission at a single excitation wavelength in two different channels, owing to the energy transfer processes within the nanoparticle. The nanoarchitecture and the presence of both Cy5.5 and Cy7 dyes provided a favorable agreement between fluorescence emission and quenching, to achieve optical imaging and photoacoustic and photothermal properties. Conclusion: We obtained rationally designed nanoparticles with outstanding stability in biological environment. At appropriate doping regimes, the presence of Cy5.5 and Cy7 dyes allowed us to tune fluorescence emission in the NIR for optical imaging and to exploit quenching processes for photoacoustic and photothermal capabilities. These nanostructures are promising in vivo theranostic tools for the near future. Keywords: dye-doped nanoparticles, optical imaging, NIR imaging, photoacoustic imaging, photothermal therapy, PEGylated nanoparticles

Sara Capolla,1 Chiara Garrovo,2 Sonia Zorzet,1 Andrea Lorenzon,3 Enrico Rampazzo,4 Ruben Spretz,5 Gabriele Pozzato,6 Luis Núñez,7 Claudio Tripodo,8 Paolo Macor,1,9 Stefania Biffi2 1Department of Life Sciences, University of Trieste, 2Institute for Maternal and Child Health – IRCCS “Burlo Garofolo”, Trieste, 3Animal Care Unit, Cluster in Biomedicine (CBM scrl), Trieste, Italy; 4Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, Italy; 5LNK Chemsolutions LLC, Lincoln, NE, USA; 6Department of Medical, Surgery and Health Sciences, University of Trieste, Trieste, Italy; 7Bio-Target, Inc., University of Chicago, Chicago, IL, USA; 8Department of Human Pathology, University of Palermo, Palermo, Italy; 9Callerio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy Abstract: The expectations of nanoparticle (NP)-based targeted drug delivery systems in cancer, when compared with convectional therapeutic methods, are greater efficacy and reduced drug side effects due to specific cellular-level interactions. However, there are conflicting literature reports on enhanced tumor accumulation of targeted NPs, which is essential for translating their applications as improved drug-delivery systems and contrast agents in cancer imaging. In this study, we characterized biodegradable NPs conjugated with an anti-CD20 antibody for in vivo imaging and drug delivery onto tumor cells. NPs’ binding specificity mediated by anti-CD20 antibody was evaluated on MEC1 cells and chronic lymphocytic leukemia patients’ cells. The whole-body distribution of untargeted NPs and anti-CD20 NPs were compared by time-domain optical imaging in a localized human/mouse model of B-cell malignancy. These studies provided evidence that NPs’ functionalization by an anti-CD20 antibody improves tumor pharmacokinetic profiles in vivo after systemic administration and increases in vivo imaging of tumor mass compared to non-targeted NPs. Together, drug delivery and imaging probe represents a promising theranostics tool for targeting B-cell malignancies. Keywords: active targeting, optical imaging, tumor accumulation

Abstract Background Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection. Results The efficacy of the active 1-35 fragment of Bac7 was assayed in serum and plasma, and its stability in biological fluids analyzed by Western blot and mass spectrometry. The ability of the peptide to protect mice against Salmonella was assayed in a typhoid fever model of infection by determination of survival rates and bacterial load in liver and spleen of infected animals. In addition, the peptide's biodistribution was evaluated by using time-domain optical imaging. Bac7(1-35) retained a substantial in vivo activity showing a very low toxicity. The peptide increased significantly the number of survivors and the mean survival times of treated mice reducing the bacterial load in their organs despite its rapid clearance. Conclusions Our results provide a first indication for a potential development of Bac7-based drugs in the treatment of salmonellosis and, eventually, other Gram-negative infections. The in vivo activity for this peptide might be substantially enhanced by decreasing its excretion rate or modifying the treatment schedule.