therapeutic nanoproducts: ejh

european journal of histochemistrya journal of functional cytology

ISSN 1121-760Xvolume 63/ supplement 1

2019

ejhunder the auspices of

the University of Pavia, Italy

Therapeutic nanoproducts: from biology

to innovative technology

19-20 June 2019

Aula PocchiariIstituto Superiore di Sanità, Rome

Co-Organized by Istituto Superiore di Sanità (ISS)

andAssociazione Italiana di Colture Cellulari (AICC)

SCIENTIFIC COORDINATORS

Stefania MeschiniMichele Caraglia

SCIENTIFIC SECRETARIAT

Maria Condello

ORGANIZING SECRETARIATAnna Tobelli

Massimo DonadelliEvelin Pellegrini

european journal of histochemistrya journal of functional cytology

ISSN 1121-760Xvolume 63/ supplement 1

2019

ejhunder the auspices of

the University of Pavia, Italy

Published by PAGEPress, Pavia, Italy

Editorial Office:Via A. Cavagna Sangiuliani 5, 27100 Pavia, ItalyPhone: +39.0382.464340 - Fax: +39.0382.34872E-mail: [email protected]

Printed quarterly by:Press Up s.r.l. via E.Q. Visconti, 90, 00193 Roma, Italy Tel. +39.0761.527351 – Fax +39.0761.527254.

Annual SubscriptionsEurope: Euro 250All other Countries: Euro 300

Subscriptions, cancellations, business correspondence andany enquiries must be sent to PAGEPress Publications,Pavia, Italy.Cancellations must be received before the end ofSeptember to take effect at the end of the same year.

No part of this publication may be reproduced, stored ina retrieval system or transmitted in any form or by anymeans (electronic, electrostatic, magnetic type, mechani-cal, photocopying or otherwise) without written permis-sion by the Publishers.

Reg. Tribunale di Pavia n. 289/23.2.1984.

Recognized by the Ministero per i Beni e le AttivitàCulturali, Italy as a publication of high cultural value.

Associato all’USPI Unione Stampa Periodica Italiana

Disclaimer. Whilst every effort is made by the publishersand the editorial board to see that no inaccurate or mislead-ing data, opinion or statement appears in this journal, theywish to make it clear that the data and opinions appearingin the articles or advertisements herein are the responsibil-ity of the contributor or advisor concerned. Accordingly, thepublisher, the editorial board and their respective employ-ees, officers and agents accept no liability whatsoever forthe consequences of any inaccurate or misleading data,opinion or statement.

Editor in ChiefC. Pellicciari

Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani” Università di Pavia

EditorsM. BiggiogeraM. Malatesta

European Journal of Histochemistrya journal of functional cytology

The European Journal of Histochemistry was foundedin 1954 by Maffo Vialli and published till 1979 underthe title of Rivista di Istochimica Normale ePatologica, from 1980 to 1990 as Basic and AppliedHistochemistry and in 1991 as European Journal ofBasic and Applied Histochemistry. It is now publishedunder the auspices of the University of Pavia, Italy. The European Journal of Histochemistry is the offi-cial organ of the Italian Society of Histochemistryand a member of the journal subcommittee of theInternational Federation of Societies forHistochemistry and Cytochemistry (IFSHC), and hasbeen an influential cytology journal for over 60 years,publishing research articles on functional cytologyand histology in animals and plants.

The Journal publishes Original Papers, TechnicalReports, Reviews, Brief Reports, Letters to the Editor,Views and Comments, and Book Reviews concerninginvestigations by histochemical and immunohisto-chemical methods, and performed with the aid oflight, super-resolution and electron microscopy,cytometry and imaging techniques; attention is alsogiven to articles on newly developed or originallyapplied histochemical and microscopical techniques.

Coverage extends to: - functional cell and tissue biology in animals and plants;- cell differentiation and death;- cell-cell interaction and molecular trafficking;- biology of cell development and senescence;- nerve and muscle cell biology;- cellular basis of diseases.

Editor in ChiefCarlo Pellicciari (University of Pavia, Italy)

EditorsMarco Biggiogera (University of Pavia, Italy)Manuela Malatesta (University of Verona, Italy)

Editorial Board

F. Cima, Padua (Italy), L. Cocco, Bologna (Italy), A.C. Croce, Pavia (Italy), G. Cutroneo, Messina (Italy),E. Falcieri, Urbino (Italy), A. Franchitto, Rome (Italy),M. Harata, Sendai (Japan), P. Hozak, Prague (CzechRepublic), Z. Kmiec, Gdansk (Poland), N.M. Maraldi,Bologna (Italy) F.J. Medina, Madrid (Spain), G. Meola,Milan (Italy), S. Modina, Milan (Italy), M. Pavelka,Vienna (Austria), M.T. Perra, Cagliari (C.A. Redi, Pavia(Italy), G. Rindi, Rome (Italy), S. Shibata, Tokyo (Japan),C. Schoeffer, Vienna (Austria)

Managing Board of the Italian Society ofHistochemistry for the years 2018-2021

Elisabetta Falcieri (President)University of Urbino “Carlo Bo”, Italy

Antonio Franchitto (vice-President)University of Rome “La Sapienza”, Italy

Francesca Cima (Member)University of Padua, Italy

Giuseppina Cutroneo (Member)University of Messina, Italy

Silvia Modina (Secretary)University of Milan, Italy

Carlo Pellicciari (past-President)University of Pavia, Italy

Editorial StaffNadia Moscato, Managing EditorCristiana Poggi, Production EditorTiziano Taccini, Technical Support

2017 Impact factor: 2.217. ©JCR Clarivate Analytics

table of contents

Therapeutic nanoproducts: from biology to innovative technology

Main Lecture ........................................................................................1

Abstracts ..............................................................................................2

european journalof histochemistryISSN 1121-760X

volume 63/supplement 12019

ejh

1

European Journal of Histochemistry 2019; vol. 63; supplement 1

MAIN LECTURE

THERAPEUTIC NANOPRODUCTS: FROM BIOLOGY TOINNOVATIVE TECHNOLOGY

S. Meschini1, M. Donadelli2, M. Condello1, E. De Smaele3, M.T. DiMartino4, E. Pellegrini1, C. Riganti5, K. Scotlandi6, F. Zazzeroni7,M. Caraglia8

1National Center for Drug Research and Evaluation, NationalInstitute of Health, Rome; 2Department of Neurosciences,Biomedicine and Movement Sciences, Section of Biochemistry,University of Verona, Verona; 3 Department of ExperimentalMedicine, Sapienza University of Rome, Rome; 4Department ofExperimental and Clinical Medicine, Magna Graecia University,Catanzaro; 5Department of Oncology, University of Turin;6IRCCS Istituto Ortopedico Rizzoli, Experimental OncologyLaboratory, Bologna; 7Department of Biotechnological andApplied Clinical Sciences, University of L’Aquila, L’Aquila; and8Department of Precision Medicine, University of Campania “L.Vanvitelli”, Naples, Italy

The main objective of every scientific progress is the improve-ment of wellness and human health. New strategies are necessaryfor safe and effective therapeutic treatments, to reduce toxicityand the side effects of conventional drugs and improve the effi-ciency and accuracy in the biological targeting. This Congress aims to give an overview of all the potential ther-apeutic and biomedical applications and the issues that must befaced when you plan to start working with nanometric dimensionmaterials.The enhancement of the therapeutic efficiency of a system withthese dimensions requires the acquisition of new skills, within astrategic planning that leads to the expansion and sharing ofknowledge in a multidisciplinary environment. The Conferencewill deepen the mechanisms of interaction between new chemi-cal-biological formulations and biophysical systems to improvethe transport of drugs in normal and tumor cells. Likewise, it isimportant to improve knowledge and encourage the optimizationof nanometric materials production methods and to discuss theregulatory aspects. Knowledge on essential questions such ascharacterization of nanomaterials, their hazards, the assessmentand management of risks is surely improved by the numerousefforts made in biomedical research and development. The pro-tection of health and environment needs to be increased by alsoimproving the current laws and regulatory system.

The conference will be divided into three sessions, with the fol-lowing titles:

1. Cell Interactions of Therapeutic Vesicles; 2. Advanced Biomedical Technology; 3. Nanosized Innovative Approaches.

The first session includes seven reports by eminent scientists onthe therapeutic use of vesicles including liposomes, micro-vesi-cles and exosomes as nanocarriers. Small membranous vesiclesare heterogeneous and play different biological functions, someof them can be shed from the surface of cells and have an impor-

tant role in mediating intercellular communication. The bidirec-tional vesicular trafficking from and to the plasma membraneand subsequent redistribution in the surface of lipids contributeto intra and extracellular large vesicle formation. Tumor derivedextracellular vesicles like exosomes can carry complex bioactivemolecules, such as nucleic acids and proteins, which can influ-ence the formation of metastases. The information contained inthe exosomes is transmitted to adjacent tumor cells and thisallows an interchange of messages between malignant cells.Moreover, these vesicles may move to distant regions of the bodyand modulate the microenvironment to form a pre-metastaticniche. Tumor derived extracellular vesicles can also be consideredas potential biomarkers for tumor development and metastasis.Chemotherapeutic drugs can be encapsulated in liposomes,sometimes to improve their uptake and minimize side effects.Drugs are encapsulated in the liposomes so increasing theantiproliferative, cytotoxic and apoptotic effect especially in theresistant cancer cells. Deepening the knowledge of the structure,role, toxicity and fate of extracellular vesicles is important forusing them as carriers to determine more specific delivery totumor sites. The second session includes lectures on the development of newapproaches for the improvement in medical and biotechnologicalapplications. Biomedical technology is the science that preciselycontrols the structure of matter at the molecular level and iswidely viewed as the most significant technological frontier cur-rently being explored in the fields of engineering and computingresearch. These technologies aim to exploit on the possibility ofmanipulating and organizing atoms or molecules in order toobtain some new medical devices that can be used to improvehuman health conditions. These devices can be integrated func-tional structures, mainly composed of mixtures of materials thatexhibit peculiar properties and capabilities that are derived fromthe spatial-temporal organization of components, as well as fromthe coordination and regulation of action of individual compo-nents and their surface properties correlated to their design. Theorganization of these components in the medical devices mayallow them to perform multistep work processes. Furthermore,many micro- and nano-devices can play a significant role inmedicine even when they are not implanted and are instead usedexternally as wearables or as laboratory devices.The third session will cover the study and use of nanostructuredmaterial in both inflammatory and cancer diseases. Topics con-cerning the use of nanocellulose, integrated microdevices, hyper-thermic nanoparticles, porous silica nanovectors and the possi-bility of using a sensitive super-resolution technique for the imag-ing of dynamic biological processes will be presented. Finally, theposition of nanomaterials in the framework of REACH(Registration, Evaluation, Authorisation and restriction ofChemicals) regulations will be discussed. REACH aims toimprove the protection of both human health and environmentthrough the better and earlier identification and evaluation of theintrinsic properties of chemical substances. It is the over-archinglegislation, which is applicable to the manufacture, placing dis-tribution on the market and use of substances on their own, inpreparations or in articles. Until now, nanomaterials are coveredonly by the definition of “substances” in REACH, but there is noexplicit reference to nanomaterials. Therefore, the understandingof how nanomaterials must be registered and regulated by lawsis relevant for both scientists and companies. This Congress will give an overview of all the potential applica-tions and problems that must be solved when we start workingwith nanometric materials.

ABSTRACTS

MICROVESICLES AND EXOSOMES IN METABOLIC DISEASES: THEIR ROLE IN INFLAMMATION

L. Dini1,2, S. Tacconi3, E. Panzarini3

1Dept. of Biology and Biotechnology “C. Darwin”, SapienzaUniversity of Rome, Rome, Italy; 2CNR-Nanotec, Lecce, Italy;3Dept. Biological and environmental science and technology,University of Salento, Lecce, Italy

E-mail: [email protected]

Multifactorial abnormalities, such as hyperglycemia, elevatedtriglyceride levels, low high-density lipoprotein cholesterol levels,etc., characterize metabolic diseases, whose progression andpathological features rely on the occurrence of local or systemicinflammation. Among immune cells, macrophages are criticaleffectors in the inflammation as diversity and plasticity are twotheir hallmarks: they can change the functional status from apro-inflammatory phenotype (classical activation called M1) toan anti-inflammatory one (alternative activation called M2) inrelation to the microenvironment and external stimuli. In thiscrosstalk, extracellular vesicles (EVs), including microvesicles(MVs) and exosomes (EXOs), play a pivotal role as they cantransport proteins and nucleic acids to adjacent cells or to dis-tant organs. In the current study, we characterized EVs shed byin vitro differentiated (M0) THP-1 human monocytes uponhyperglycaemic stress obtained by treating M0 macrophageswith 15 and 30 mM glucose for 24 h, by using transmission andscanning electron microscopy and dynamic light scattering(DLS) analysis. Moreover, we evaluated the ability of EVsreleased by glucose stressed cells to interfere with macrophagespolarization by culturing differentiated THP-1 cells for up to 24hin the presence of MVs, EXOs and in the conditioned medium-CM without EXOs and MVs of M0 glucose stressedmacrophages. Genes (IL-1β, IL-6, IL-10, IFN-α, TNF-α, CCL17,TLR4, NFkB) and proteins (iNOS, CD86, CD163, MHC-I, STAT6)expression was used to detect the markers of macrophage polar-ization. Results showed that in hyperglycemic condition, that is astrong inducer of the pro-inflammatory markers, M0macrophages are induced to an increased secretion of EVs, inparticular of microvesicles that, in turn, enhance the M0 polar-ization towards a pro-inflammatory phenotype. This opens newperspectives in understanding the role of macrophages in dia-betes and in the cell-cell communication based on EVs releaseand provide potential therapeutic targets for the treatment ofinflammatory conditions during metabolic diseases.

INSIGHT INTO SURFACTANT-BASED “SOFT” NANOCAR-RIERSM. Carafa

Department of Drug Chemistry and Technology, SapienzaUniversity of Rome, Italy


The application of nanotechnology in diagnosis and therapy iswidely accepted as a potential driver of medical innovation.During the last decades, the formulation of surfactant-based“soft” nanocarriers (micelles, micro and nanoemulsions, nio-somes), as a tool to improve drug delivery,brought an everincreasing interest among the scientists working in the area ofdrug delivery systems. In particular, niosomes1,2 are self assem-bled vesicular nanocarriers obtained by hydration of syntheticsurfactants and appropriate amounts of cholesterol or otheramphiphilic molecules. To get insight into the formulation of sur-factant-based “soft” nanocarriers to develop well definednanoformulations of therapeutic relevance an adequate charac-terization is of paramount importance. Determination of physico-

chemical features such as particle size and morphology, surfacecharge and stability are indispensable to better evaluate nanocar-rier proper application. Furthermore, better understanding of theextra- and intra-cellular fate of the nanocarriers in relation withbiological environments in vitro and in vivo is essential to under-stand how dynamic interactions with biological components altertheir fate and performance in vivo3. Nevertheless, the opportuni-ties of nanotechnologies in the health sector are accompanied bychallenges in the regulation of these products4. Sufficient knowl-edge on their quality, safety, and efficacy has to be gained to sup-port the regulatory decision to allow the translation towardsclinical applications. The highly interdisciplinary nature of thisfield, from basic chemistry and physics to clinical evaluations,makes researches in developing nanomedicines very fruitful. Thismeans there are still many open challenges that need to be over-come and there are still opportunities for academic and industri-al scientists to make a decisive impact.

References1. Marianecci C, et al. Adv Colloid Interface Sci 2014;205:187-206.

2. Moghassemi S, Hadjizadeh A. J Contr Rel 2014;185:22-36.

3. Seo SJ, et al. Nano Today 2017;14:84-99.

4. Bremer-Hoffmann S, et al. J Interdisc Nanomedicine 2018;3:4-15.

MESENCHYMAL STROMAL FREEZE-DRIED EXTRACEL-LULAR VESICLES FOR NANOMEDICINE: FROM GMP-COMPLIANT PRODUCTION TO NEXT GENERATION DRUG DELIVERY SYSTEMS. Perteghella, E. Bari, M.L. Torre

Department of Drug Sciences, University of Pavia, Italy


Mesenchymal Stem Cells (MSCs)-secretome, composed of solu-ble proteins and extracellular vesicles (EVs), represents a newtherapeutic approach for chronic and acute diseases in place ofparental cells. A perspective view is here provided about thestate-of-art and the missing steps required for the secretome“pharmaceuticalization” into a high quality, safe and effectivemedicinal product, as well as for optimal formulation. A scalableand GMP-compliant production process is the first requirementand it is now established that the ultrafiltration is the only onethat fit. Stability and shelf-life are the second issue: freeze-dryingis the technological process widely used in pharmaceutical field,which is best suited to both protein and EVs. We combined ultra-filtration and lyophilisation techniques obtaining a ready-to-usepowder, lyo-secretome1-3. The process did not affect EV integrityand morphology, and allowed to obtain a safe and effective prod-uct, which can be considered as a new Active PharmaceuticalIngredient (API). Efficacy of lyo-secretome was proven againstoxidative stress-induced damages2, as immunomodulant agent3and in a wound healing murine model. Furthermore, proteomiccharacterization of lyo-secretome revealed that it containsalpha1-antitripsin (AAT) with in vitro anti-elastase activity,paving the way for the use in lung degeneration AAT deficiency.At the same time, characteristics of EVs make them ideal candi-dates as drug delivery systems (DDS), due to their high stabilityin bloodstream and low immunogenicity. Moreover, the innatehoming ability to injured site, make EVs suitable smart DDS toavoid off side effects, and enhance the specific uptake by targetcells. In this context, EVs can also be used as “carrier in carrier”device constituted by EVs containing nanosystems loaded ofdrugs4. These results demonstrate that MSC-secretome can beused both as API and DDS; although human and veterinary clin-ical trials are still few, we are confident that many will start inthe near future.

References1. Perteghella et al. PCT/IB2017/056591;2016.

2. Bari E, et al. Cells 2018;7. pii:E190.

2

Therapeutic nanoproducts: from biology to innovative technology (ISS - AICC)

3. Bari E, at al. Nanomedicine 2019;14:753-65.

4. Perteghella S, et al. Int J Pharm 2017;520:86-97.

NANOMEDICINE IN CANCER: TOWARD OVERCOMINGCHEMORESISTANCE

M. Caraglia1, C. Leonetti2, C. Riganti3, V. Campani4, S. Zappavigna1, M. Porru2, J. Kopecka3, M. Abate1, G. De Rosa1

1Department of Biochemistry, Biophysics and General Pathology,University of Naples “Vanvitelli”, Naples; 2UOSD SAFU, IRCCSRegina Elena National Cancer Institute, Rome; 3Department ofOncology, University of Turin, Turin; and 1Department ofPharmacy, Federico II University of Naples, Naples, Italy


Intrinsic and acquired drug resistance (multidrug resistance orMDR) is a major challenge in treating cancer patients. One ofthe most frequent cause of chemoresistance is the elevatedexpression levels of drug efflux pumps, such as P-glycoprotein(Pgp) belonging to the ATP-binding cassette (ABC) superfamily.Nanomedicine represents a promising tool to overcome MDR atdifferent levels. First of all, nanovectors have been proposed todeliver ABC inhibitors co-administered or co-encapsulated withchemotherapeutics. Molecules with a well-established activity ofABC-inhibitors, such as verapamil, have showed a systemic toxi-city, while alternative strategies to inhibit ABC expression areemerging e.g., with the bisphosphonates as zoledronic acids(ZOL). Recently, we demonstrated that the use of nanovectors,such as liposomes and self-assembling nanoparticles (SANPs)can be used to deliver ZOL in tumors1,2. We also observed thatZOL, only by using nanovectors, reduces the expression of Pgp inMDR cancer cells3,4, thus restoring the sensitivity of MDRtumors to chemotherapeutics such as doxorubicin or carbo-platin4. More recently, chitosan-based nanoparticles have beendeveloped to co-encapsulate ZOL and doxorubicin for combinedtherapy against MDR tumors5. In this case, also a synergisticinhibition activity was observed when combining ZOL and dox-orubicin on MDR cells5. A second level in which thenanomedicine can be used to overcome the MDR is the deliveryof nucleic acids such as small interfering RNA (siRNA) ormicroRNA (miRNA). Indeed, siRNAs can be used to inhibit theexpression of a protein involved in MDR, while miRNAs areimportant modulators or protein expression and their mis-expression has been correlated to the occurrence of MDR. Thus,polymeric micelles have been developed for the co-delivery of ananti-survivin siRNA and paclitaxel for the reversal of drug resis-tance in tumors6. On the other hand, SANPs encapsulating themiR603 have been proposed to restore sensitivity to temozolo-mide in glioblastoma cells (unpublished data). Finally, a thirdlevel of attention should be paid on the biomaterials used whenusing nanovectors in MDR tumors. The use of liposomes canovercome the resistance to doxorubicin in cells overexpressingPgp. In addition, the use of suitable biomaterials, and in partic-ular the inclusion of PEGylated lipid into the formulation, can beparticularly useful to inhibit activity of the efflux pumps.

References1.Salzano et al. 2011. 2.Marra et al. 2012

3.Kopecka et al. Oncotarget – 2015

4.Kopecka et al. Oncotarget – 2016

5Giarra et al. Pharmaceutics 2018

6.Salzano G, et al. Mol Cancer Ther. 20157.Kopecka et al. Nanomedicine 2014

HACKING PANCREATIC CANCER DRUG RESISTANCEWITH NOVEL NO-GEMCITABINE PRODRUGS ENCAPSU-LATED IN LIPOSOMES F. Masetto1, K. Chegaev2, E. Gazzano3, N. Mullappily1,B. Rolando2, R. Fruttero2, S. Arpicco2, C. Riganti3, M. Donadelli1

1Department of Neurosciences, Biomedicine and MovementSciences, University of Verona; 2Department of Drug Science andTechnology, University of Turin; 3Department of Oncology,University of Turin, Italy


Since the late 1990’s, gemcitabine (GEM) has been available asthe standard of care regimen for advanced pancreatic ductaladenocarcinoma (PDAC). However, the resistance to chemother-apy treatment is the main issue against a successful anti-cancertherapy.1 Nitric oxide (NO) is a small free radical moleculeinvolved in many physiologic and pathologic pathways in humancells. It plays also a key role in tumor biology and at micromolarconcentrations it displays antitumor effects.2,3 In this study, wesynthesized a library of seven NO-releasing gemcitabine conju-gates (NO-GEMs) in order to improve the effectiveness of GEMin PDAC cell lines. We used as a cellular model of PDAC Panc1and MiaPaca2 cell lines, which were previously demonstrated tobe more resistant or sensitive to the GEM treatment, respective-ly. Among the new NO-GEM prodrugs, we identified GCB2 as themost effective compound in inhibiting cancer cell growth. Toimprove its uptake and minimize side effects we encapsulated itin liposomes (LIPO-GCB2), increasing its antiproliferative andapoptotic effect especially in GEM-resistant Panc1 cells, as com-pared to standard GEM. We observed that the multidrug resist-ant (MDR) family member MRP5, which is involved in the cellu-lar export of GEM, is more expressed in GEM-resistant than inGEM-sensitive PDAC cell lines. Notably, LIPO-GCB2 inhibitedMRP5 activity by nitrosylation, favoring the intracellular accu-mulation of GEM and triggering apoptotic cell death in GEM-resistant PDAC cells. Our results support the development of anew anti-tumoral strategy to efficiently affect GEM-resistantPDAC cells based on the usage of NO-releasing GEM encapsu-lated in liposomes.

References1. Binenbaum Y, Na’Ara S, Gil Z. Drug Resist Updat 2015;23:55-68. 2. Huerta S, Chilka S, Bonavida B. Int J Oncol 2008;33(5):909-927.3. Riganti C, et al. Cancer Res 2005;15;65:516-25.

LIPOSOMAL CARRIER AS POSSIBLE THERAPEUTICVESICLES: MORPHO-FUNCTIONAL INVESTIGATIONM. Battistelli

Department of Biomolecular Sciences (DiSB), University ofUrbino Carlo Bo, Italy


In the last decades, deficiency of macro- and micronutrients wasconsidered as a serious problem associated with the increase inthe human population. Micronutrient malnutrition causes blind-ness and anemia (even death) in more than half of the world’spopulation, especially among women of reproductive age, preg-nant and lactating women, and preschool children. In particular,iron deficiency is one of the most frequent health problems,affecting approximately one third of the entire population. Itsrequest may not be satisfied with a regular diet, due to a low ironbioavailability and to dietary factors that limit its absorption.Iron supplementation is considered a global economic and effec-tive strategy for prevention and control of anemia, in particular,during pregnancy1. Therefore, there is the need to develop newexperimental protocols to improve its bioavailability. Liposomes2are vesicular structures with double-layer lipid membrane, whichare proved particularly interesting in the biomedical field as adelivery system of pharmaceutically active substances3. In thisstudy, ultrastructural analyses have been carried out to verify if

3


liposome dehydration process induces structural alterations oflipid membranes, which could compromise carrier integrity andfunction. Morphological integrity of Biofer and Lifervit (two newcommercial iron carriers), both in liquid form and in dried pow-der, has been investigated by means of transmission electronmicroscopy4. Moreover, liposome interaction with U937 cell line,a well-known phagocytosis human model, has been studied. Bothcompounds revealed a good stability and the ability to penetrateinto cells, interacting with cytoplasmic organelles without induc-ing, at least apparently, any ultrastructural damage and toxicity.Therefore, Biofer and Lifervit do not cause cellular damage, andfor that they can be considered potential candidates in ironvehiculation. Further studies are in progress to evaluate theirinteraction with intestinal cell models.

References1. Kamau MW, et al. BMC Public Health 2018;18:580. 2. Tamjidi F, et al. Innov Food Sci Emerg Technol 2014;26:366-74. 3. Keshari SA, et al. Pharmanest 2014;5:2019-33. 4. Battistelli M, et al. Microsc Res Tech 2018;81:1295-1300.

IMMUNOTOXICITY ASPECTS IN THE DEVELOPMENTAND REGULATORY EVALUATION OF NANODRUGS G. Di Felice, B. Barletta, C. Butteroni, S. Corinti

National Center for Drug Research and Evaluation, IstitutoSuperiore di Sanità, Rome, Italy


Innovative nanotechnology-based medicinal products areincreasingly developed for both therapeutic and diagnostic uses.The challenge for regulatory agencies is to ensure the properevaluation of the quality, safety, and effectiveness of nanomedi-cines undergoing clinical development and market authorization.For a safe translation into clinical applications, proper regulato-ry pathways and standardised test methods must be available.The primary function of immune system is to protect the hostfrom foreign invaders, by integrating the actions of its innate andadaptive compartments. Upon the intentional delivery of nano-drugs or diagnostic/imaging systems, they interact with immunecells that are abundant in the tissues close to the entry sites aswell as in the bloodstream. Because of nanodrugs interactionwith the immune system, the assessment of their impact in termsof immunotoxicity, and of their potential to modulate its function(suppression, stimulation, and hypersensitivity) deserves ade-quate attention in the regulatory risk-benefit assessment1. Untilnow, no specific regulatory documents have been developed toassess the immunotoxicity of nanodrugs, which are currentlyevaluated on the basis of guidelines, such as the ICHS8 Note forGuidance on Immunotoxicity Studies for HumanPharmaceuticals2, in force for conventional medicinal products.The crucial question is whether the set of first- and second-leveltests prescribed by these guidelines is capable of adequately eval-uating the immunotoxic profile of complex and broadly heteroge-neous nanodrugs that result from the combined effects of theactive pharmaceutical component, the nanocarrier, any potentialcoating, and the final formulation3. To address this question, wedeveloped a panel of in vitro assays on immunocompetent celllines to evaluate cytotoxicity, functional activation, production ofeffector molecules (Nitric Oxide, NO) and proinflammatorymediators (IL-6 and TNFα cytokines). Two cell lines of murineorigin, RAW264.7 and MH-S, have been selected being represen-tative of different exposure conditions and entry site delivery,since they belong to the monocyte-macrophage lineage from thesystemic circulation and the lung alveolar district, respectively.The results obtained in these simple and reproducible systemssupport their predictivity and potential application both to thesafe-by-design development of nanodrugs and to the implemen-tation of nano-specific regulatory guidelines.

References

1. Dobrovolskaia MA, et al. Toxicol Appl Pharm 2016:299:78-89.2. http:/ /www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002851.pdf

3. Giannakou C, et al. Int J Nanomedicine 2016;11:2935-52.

NANOSTRUCTURED MATERIALS AND DEVICESM. Grigioni1, B. De Berardis1, G. D’Avenio1, C. Daniele1, G. Bozzuto2, M. Condello2, S. Meschini2, A. Molinari2, R. Pecci1,R. Bedini1, F. Superti1

1National Center for Technological Innovation in Public Health;2National Center for Drug Research and Evaluation, IstitutoSuperiore di Sanità, Rome, Italy


The increasing diffusion of nanomaterials (NM) in the domain ofmedical devices (MD) is due to the peculiar characteristics ofsuch materials (e.g., the tunability of their physico-chemicalproperties as a function of their size), which allow to obtain ahigh biocompatibility and, at the same time, to exploit their highsurface/volume ratio. Clearly, the associated risks must carefullybe considered, besides their advantages. The need for regulatoryoversight of NM-containing DM is reflected by the new MDRegulation: MD incorporating or consisting of NM are in classIII, unless the NM is encapsulated or bound in such a mannerthat it cannot be released into the patient’s or user’s body. Inorder to identify possible mechanisms of cell-NM interaction, thecell damage potential of some relevant NM (ZnO), already usedin the fabrication of MD, was assessed by means of differenttechniques (MTT, electric cell-substrate impedance sensing -ECIS, SEM, TEM,…). Moreover, we selected a nanostructuredwound dressing product containing Ag NM already used in thefabrication of MD. The cell damage potential of these NMs wasassessed, again using different techniques. The guidance for deal-ing with NM-associated risks in the European regulatory frame-work was investigated. Standard testing on the final productmust be considered, besides the typical verification activities ofthe Notified Bodies (third part entities for conformity assess-ment). The standard cytotoxicity assays showed a good agree-ment with ECIS, which gives a continuous real-time measure-ment of cell viability, and provides valuable information on celldamage mechanisms. The results were very sensitive to NM con-centration, showing the necessity of defining clear thresholds foreach NM and contact type. In particular, it is generally agreedthat the ISO 10993 series must be revised in view of the speci-ficity of NM. Methodological suggestions have been provided in1,stating that “it is likely that the various assays as described inthe ISO 10993 series are not always appropriate as such in thetesting of nanomaterials.” Notably, the accuracy of traditionalcytotoxicity tests may fail, due to the interaction between NMsand assay materials. In our experience, a satisfactory agreementwas found between traditional and innovative (ECIS) tests. Acautionary approach about nanostructured MDs is suggested bythe new EU regulatory framework, in view of the still incompleteknowledge about them.

Reference1. ISO/TR 10993-22:2017 Biological evaluation of medical devices —Part 22: Guidance on nanomaterials

4


POLYMERIC NANOPARTICLES AS VERSATILE DRUGDELIVERY SYSTEMSD. Cosco

Department of Health Sciences, University of Catanzaro “MagnaGræcia”, Campus Universitario “S. Venuta”, Catanzaro, Italy


Polymeric nanoparticles have been extensively investigated asdrug delivery systems due to their technological features. Theycan be classified into two types based on their morphology andthese are capsules, which are systems made up of a drug-contain-ing core surrounded by a polymeric shell, and spheres, usuallycharacterized by a matrix in which the active compounds arecontained or adsorbed. In particular, aqueous-core polymericnanocapsules are used to modify the biopharmaceutical proper-ties of water-soluble drugs, thus increasing their plasmatic half-life and pharmacological efficacy besides decreasing their sideeffects. For example, novel aqueous-core polylactic acid (PLA)nanocapsules able to retain hydrophilic active compounds havebeen developed and their physico-chemical properties evaluatedas a function of the components used. The peculiar structure ofthese nanosystems favoured a massive encapsulation of antitu-mor drugs increasing their in vitro and in vivo anticancer efficacywith respect to their free forms1. The rigid shell of PLA nanocap-sules allows the efficient entrapment and physical retention ofgenetic material. For instance, plasmids and siRNAs have beenefficiently encapsulated and protected from degradation, and theresulting nanoplexes have evidenced good cell transfection fea-tures2. The polymeric nanosystems also permitted the efficiententrapment of meglumine antimoniate (glucantime), increasingits accumulation in macrophages and thus its pharmacologicalactivity against Leishmania tropica3. Another interestingbiopolymer used to obtain nanosystems is zein, a naturalhydrophobic protein belonging to alcohol-soluble prolamine-richcompounds abundantly contained in corn. Zein nanoparticleswith a mean diameter of 100-150 nm have been developed byusing various surfactants. These systems are characterized by agreat degree of stability and are able to retain various activecompounds4. The results evidence the potential clinical applica-tion of these polymeric formulations as novel drug delivery sys-tems.

References1. Cosco D, et al. Eur J Pharm Biopharm 2015;89:30-39.2. RM2013A000635.3. RM2014A000252, PCT WO2015/177820.4. Gagliardi A, et al. Int J Nanomedicine 2018;13:601-14.

MESOPOROUS SILICA-BASED NANOSYSTEMS AS AVEHICLE FOR TARGETED DELIVERY OF THE ANTINEO-PLASTIC DRUG BORTEZOMIBA. Leggio1,2

1NanoSiliCal Devices s.r.l., University of Calabria, 87036Arcavacata di Rende, Italy; 2Department of Pharmacy, Healthand Nutritional Sciences, University of Calabria, Arcavacata diRende, Italy


Traditional cancer therapies are often limited by the lack ofselectivity to cancer cells as they frequently display unwantedsevere toxicities on the healthy ones. Pharmacological propertiesof conventional anticancer drugs can be improved through theuse of drug delivery systems (DDS) composed of biocompatiblenanomaterials that can deliver therapeutic agents selectively tocancer cells while sparing normal tissues and minimizing side

effects1. The intrinsic properties of mesoporous silica nanoparti-cles (MSN) make them ideal delivery platforms2. Their structuraland chemical features as high surface area, tunable size, and easysurface modification, permit to design versatile nanosystems ableto encapsulate a variety of therapeutic agents and to host target-ing molecules able to interact selectively with specific membranereceptors overexpressed in tumor cells. A MSN-based device,FOL-MSN-BTZ, for pH-sensitive release of the antineoplasticdrug bortezomib (BTZ) in cancer cells over-expressing folatereceptor (FR) has been developed. It consists of a nanometrichoneycomb-like silica frame that bears on the external surfacefolic acid (FOL) as targeting function (folate receptor specificligand) and in the inner pores the antineoplastic drug BTZ linkedby means of a pH-sensitive bond. The boronic acid group of BTZinteracts with diol functionalities on the MSNs pore surface byforming cyclic boronic esters that are quite stable and arehydrolysed only in acidic environments, such as those of lyso-somes and endosomes once target tumour cells are reached; thelower pH encountered therein triggers the drug release. FOL-MSN-BTZ was tested on cancer cells overexpressing the folatereceptor (FR+ cells) and on FR normal cells, in order to inves-tigate MSNs behavior. FOL-MSN-BTZ was able to induce deathexclusively in FR+ tumor cells, while free BTZ resulted toxic forall cell lines tested, including FR normal cells.

References1. Peer D, et al. Nat Nanotechnol 2007;2:751-60.2. Argyo C, et al. Chem Mater 2014;26:435-51.

MESENCHYMAL STEM CELLS AS A VEHICLE FOR MUL-TIMODAL NANOPARTICLESE. Martella1, B. Dozza2, C. Bellotti3, S. Lenna3, M. Columbaro3, A. Guerrini1, C. Ferroni1, G. Sotgiu1, L. Cevolani3, E. Lucarelli3, G. Varchi1, D.M. Donati2,3, S. Duchi1

1ISOF-CNR, Bologna; 2Dept Biomedical and NeuromotorSciences, University of Bologna; 3IRCCS Istituto OrtopedicoRizzoli, Bologna, Italy


In the past decade, the use of human Mesenchymal Stem Cells(hMSC) as cell based drug delivery vectors to treat cancer, hassignificantly developed. hMSC display the capacity to internalizeand retain drugs and nanoformulations1, and they can infiltratethe tumor stroma via cytokines-driven clues released from thetumor2. Osteosarcoma (OS) is the most common bone cancerdiagnosed in children and adolescents. The development ofchemoresistance and lung metastasis limits the success rate to aworldwide 5-years patients survival rate of 70%. Therefore, OStherapy might be moving toward nanotechnology-based drugdelivery systems (DDS) to reduce the cytotoxicity of antineoplas-tic drugs and improve their pharmacokinetics3. As well, photody-namic therapy (PDT) represents an approach to kill OS cells cir-cumventing chemoresistance4,5. The aim of this study is to engi-neer hMSC carrying DDS (hMSC-DDS) for OS treatment, usinga combination of PDT (Chlorin e6 as photosensitizer) andchemotherapy (Paclitaxel-PTX, as antineoplastic) and test itsefficacy in a 3D-spheroid OS model. DDS were prepared fromhigh molecular weight and hydrosoluble keratin functionalizedwith Ce6 and loaded with PTX to obtain PTX-Ce6@kernanoparticles (KNPs)6. Our results show that hMSC efficientlyinternalize KNPs after 24 h and retain them intracellularly forseveral days in culture. The migratory capacity is not impaired byPTX internalization, and in the 3D model, the cell death inducedby PTX-cell cycle blockage and light irradiation is 95%. Ourdata corroborate the use of MSCs-DDS as an high effectivetreatment in a 3D tumor model, and set the basis for the preclin-ical translation. If proved to be effective in vivo, this approach

5


could revolutionize OS pharmacological treatment in affectedpatients.

References

1. Duchi S, et al Bioconjug Chem 2014;12:649-55.

2. Studeny M, et al Can Res 2012;62:3603-8.

3. Patra JK, J Nanobiotechnology 2018; 16(1):7.

4. de Miguel GC, et al. Photodiagnosis Photodyn Ther 2018;21:79-85.

5. Duchi S, et al. J Control Release 2013;168:225-37.

6. Martella E, et al. Int J Mol Sci 2018,19:3670.

VITVO: MIMICKING IN VIVO COMPLEXITY BY THEINNOVATIVE 3D MODELO. Candini1, G. Grisendi1, E.M. Foppiani1, M. Brogli1, B. Aramini2,V. Masciale3, C. Spano1, T. Petrachi4, E. Veronesi4, P. Conte5, G. Mari1, M. Dominici1,3

1Rigenerand srl, Medolla, Modena; 2Division of Chest Surgery,3Division of Oncology, Department of Medical and SurgicalSciences for Children & Adults, University-Hospital of Modenaand Reggio Emilia, Modena, Italy; 4Technopole of Mirandola,Mirandola, Modena, Italy; 5Department of Surgery, Oncology andGastroenterology, University of Padua, Istituto Oncologico VenetoIRCCS, Padua, Italy

Three-dimensional (3D) cell culture models can provide physio-logically relevant information compared with the traditional two-dimensional (2D) cell cultures, generating interest in drug dis-covery to predict response to drugs and putative compounds, alsoutilizing a personalized approach1,2. Several 3D cell culture tech-nologies have been developed to better mimic in vivo complexity,however, simple and reproducible systems able to rapidly rebuildtissue ex vivo in 3D are needed to overcome some limitations andoffer novel possibilities3–5. We developed an isolated “smart-phone-like” 3D cell culture bioreactor that can be loaded withtumor and/or normal cells in combination, which can be moni-tored using a variety of read-outs. This biocompatible device,called VITVO, sustained a 3D growth of several tumors, such aspancreatic and breast adenocarcinoma, sarcoma and glioblas-toma. Cells repopulated the 3D matrix inner core, which is com-pletely separated from the outer space by two gas-permeablemembranes and can be monitored in real-time using both fluores-cence microscopy and luminometry, even after transportation.The use of VITVO in a variety of assays focusing on oncology isreported and the efficacy of chemotherapy, biologics and cell-based anti-cancer agents was tested by comparing VITVO withan in vivo preclinical xeno-transplant model. Notably, the systemwas challenged using primary tumor cells harvested from lungcancer patients as an innovative predictive functional assay forcancer responsiveness to a checkpoint inhibitor, such as nivolum-ab. The novel features as well as the flexibility offered by thisplatform within the different approaches suggest the potential ofVITVO as an innovative 3D in vitro model for pre-clinical testingwith a possible relevant impact in other areas beside oncologyfield.

References1. Antoni D, et al. Int J Mol Sci 2015;16:5517-27.

2. Fitzgerald K A, et al. J Control Release 2015;215:39-54.

3. Shin, C S, et al. Mol Pharm 2013;10:2167-75.

4. Fischbach C, et al. Nat Methods 2007;4:855-60.

5. Horning J L, et al. Mol Pharm 2008;5:849-62.

SEC MEETS TRPS: AUTOMATED ISOLATION AND HIGH-PRECISION ANALYSIS OF EXTRACELLULAR VESICLES C. Roesch1, A.K. Pal2, S. Harrel3, R. Vogel2, M. Broom2

1Izon Science Europe Ltd, Lyon, France; 2Izon Science Ltd,Medford, MA, USA; 3Izon Science Ltd, Christchurch, NewZealand


Extracellular Vesicles (EVs) derived from biological fluids pos-sess extensive heterogeneity with regards to size, number, mem-brane composition and cargo. Tremendous research interestexists towards development and use of EV fraction of biofluidsas rich sources of diagnostic and prognostic biomarkers. Highprecision fractionation of the nano-biological content of biofluidscan dramatically reduce background, increase purity, and informon the biology of the biomarkers and therapeutic biomolecules.Size exclusion chromatography (SEC) is the most standardiz-able technique, already widely used for the purification of EVsfrom biofluids.1 Significant improvement to the use of SEC ispossible through automation and precision. Here, we developed arange of SEC columns of various sizes, with 2 resin types, sepa-rating down to 35 nm or 70 nm. We also developed a low-costprototype automatic fraction collector (AFC) that adds high pre-cision, improves repeatability, speeds up workflow. RFID tags areproposed to ensure high quality of data capture and transfer.Moreover, Tunable Resistive Pulse Sensing technology was usedfor accurate, high-resolution particle analysis (size, size range,concentration, and electrophoretic mobility) and normalization2.SEC columns provide a convenient, reproducible, and highlyeffective means of eliminating >99% of non-vesicular proteinfrom biological fluid samples, and separating exosomal and non-exosomal volumes for further downstream analysis. 35 nm poresized SEC gel leads to increased resolution, higher yield and onefraction earlier elution of EVs from plasma compared to the 70nm pore size. Use of AFC allowed precise massbased measure-ments and tunability within 30 μL of volume exiting the column.Most importantly, due to the additional functionality provided byAFC, the EV field needs to revisit the way that fraction numbers,post-SEC are used. That will be replaced with a more logicalframework, wherein the void volume is measured and disposed of,and precise volumes are used instead of the somewhat arbitraryfraction numbers. Thus, the qEV-AFC platform allows for QA,high-precision EV volume collection and minimizes samples pro-cessing related reproducibility issues for clinical studies.

References1. Boing AN, et al. J Extracell Vesicles 2014;3:23430.

2. Pedersen S. ISEV 2015 meeting, Washington DC.

NANOTECH 5.0: FROM BIOLOGY TO VIRTUAL REALITY,FAST FORWARDM. Mariani1, L. Clario2, V. Brambilla1

1Media System Lab S.r.l., Macherio (MB), Italy; 2Nanolive SA,Ecublens, Switzerland.


By combining holography with tomography, Nanolive technologyis an example of emerging smart nanoscopic technique. The 3DCell Explorer microscope guarantees three-dimensional high spa-tio-temporal resolution imaging of transparent unlabelled speci-mens, with the advantage over fluorescence techniques of notrequiring sample labelling, thus reducing potential damages toliving cells. Thanks to the absence of photo-toxicity and the incu-bated controlled environment, cells can be indeed cultured andmonitored in continuous time-lapse for days. Moreover, based onthe high sensitivity in Refractive Index measurements, the systemenables for high resolution visualization of intracellular com-partments (i.e., mitochondria, lipid droplets, lysosomes). Offeringan optimal combination between sample health, spatiotemporal

6


resolution and signal-to-noise ratio, holotomography representsnowadays one of the best solutions for long term live cell imagingapplications.

NANOCELLULOSE AND ITS COMPOSITES IN BIOMEDI-CAL DEVICESC.A. Maestri1, N. Ferrari1, S. Meschini2, E. Pellegrini2, M. Condello2, M. Scarpa1

1Department of Physics, University of Trento, Povo-Trento; and2National Center for Drug Research and Evaluation, IstitutoSuperiore di Sanità, Rome, Italy


Nanosized cellulose (NC) is a material of natural origin obtainedby an eco-friendly procedure, in the form of fibrils (NCF) or crys-tallites (CNC). In particular, CNC prepared by carboxylation andultrasonic dispersion according to Saito et al. 1, are rod-shaped,rigid and regular nanostructures with thickness 3-5 nm and 250-500 nm length2,3. The high surface area together with the stronghydrogen bonding and cation coordination capacity, favour theinteraction of CNCs with the surrounding species. Accordingly,CNCs are easily dispersed in water, while undergo a layer-by-layer assembly when dried and entangle in soft hydrogels by acation-driven process. CNC-derived materials show unique fea-tures in term of structure and functional properties and are suit-able for the fabrication of medical and life science devices.However, the nanotoxicity of CNC as component of biomedicaldevices is still an open question. In this presentation we willreport about the fabrication of CNC hydrogels and their incorpo-ration in nanocomposite materials. The material properties suchas microstructure, mechanical performances, and in vitro toxicityare also presented. References1. Isogai A, et al. Nanoscale 2011;3:71-85.

2. Maestri CA, et al. Mater Chem B 2017;5:8096-104.

3. Maestri CA, et al. Sci Rep 2017;7:11129.

NANOCARRIERS FOR NEUROMUSCULAR DISEASESM. Costanzo1, F. Carton1,2, V. Guglielmi1, L. Calderan1, M. Repellin1,2*, G. Lollo2, I. Andreana3, S. Arpicco3, B. Stella3, M. Malatesta1

1Dept. of Neurosciences, Biomedicine and Movement Sciences,University of Verona, Italy; 2University of Lyon, Université ClaudeBernard Lyon 1, CNRS, LAGEPP UMR 5007, France; 3Dept. ofDrug Science & Technology, University of Turin, Italy


Myotonic dystrophy type 1 and 2 (DM1 and DM2) are geneticdisorders caused by CTG and CCTG expanded repeats, respec-tively. The transcribed expanded RNAs accumulate in nuclearfoci where splicing factors are sequestered, thus causing a gener-al splicing deregulation in multiple tissues. In both DMs, patho-logical traits are muscle weakness, dystrophy/atrophy andmyotonia, with disabling effects and possibly premature death.Currently, no therapy is available for DMs. Different therapeuticmolecules have been successfully tested in experimental modelsto target the expanded RNAs; however, most of the delivery ortoxicity problems remain unsolved. Our research aims at design-ing a novel therapeutic strategy using biocompatible nanoparti-cles (NPs) to administer drugs or oligonucleotides able to coun-teract RNA toxicity in DMs, with special reference to skeletalmuscle. Drug-loaded NPs may improve therapeutic efficacy byprotecting the encapsulated molecules from enzymatic degrada-tion and ensuring their delivery and sustained release inside thecell. Among the different NPs we tested, poly(lactide-co-glycol-

ide) (PLGA) NPs and hyaluronic acid (HA)-based NPs proved tobe highly biocompatible for cultured murine and humanmyoblasts and myotubes1,2. We elucidated the mechanisms oftheir uptake, distribution and degradation by fluorescence andtransmission electron microscopy. We also investigated NPs dis-tribution in explanted mouse skeletal muscles maintained underfluid dynamic conditions3. Both PLGA and HA NPs proved toefficiently load pentamidine, one of the promising therapeuticagents, and preliminary results on cultured myoblasts from DM1patients demonstrated that pentamidine delivered by PLGA NPsreduced the pathological accumulation of nuclear foci with lim-ited cytotoxic effects.

*INVITE project received funding from EU Horizon 2020 GANo 754345.

References1. Costanzo M, et al. Nanomedicine (Lond) 2019;14(3):301-16.2. Guglielmi V, et al. Int J Pharm 2019 ;560:347-56.3. Carton F, et al. Eur J Histochem 2017;61(4):2862.

HYPERTHERMIC NANOPARTICLES TO TRIGGER LIPOLYSISL. Calderan1, F. Vurro1, S. Mannucci1, F. Boschi2, S. Tambalo3, P. Lievens1,D. Prosperi4, M. Malatesta1

1Department of Neurosciences, Biomedicine and Movement,Anatomy and Histology Section, University of Verona; 2Department of Computer Science, Università di Verona; 3Centerfor Neuroscience and Cognitive Systems @UniTn, IstitutoItaliano di Tecnologia, Rovereto; and 4Department ofBiotechnology and Biosciences, University of Milano-Bicocca,Italy


During last years, evidence has been provided on the involvementof obesity in the pathogenesis and aggravation of several life-threatening diseases. In this view, we set up an innovative proto-col to induce a nanoparticle-mediated lipolysis in vitro by com-bining light and electron microscopy, and biochemical approach-es. Under appropriate administration conditions, 3T3-L1 mouseadipocytes proved to efficiently and safely internalize superpara-magnetic iron oxide nanoparticles (SPIONs), which are able toproduce heat when subjected to alternating magnetic field1-3.Thus, 3T3-L1 adipocytes were submitted to SPIONs-mediatedhyperthermia treatment (SMHT), with the aim of modulatingtheir lipid content4,5. The treatment resulted in a significant delip-idation persisting for at least 24 h, in the absence of cell death,damage or dedifferentiation. Interestingly, some factors normallylinked with lipolysis event or in lipid metabolism6 were not mod-ulated upon SMHT, suggesting the involvement of a novel/alter-native mechanism in the lipolysis observed. Notably, the sameSMHT was able to induce delipidation also in primary culturesof human adipose-derived adult stem cells. The success of thisnew approach in vitro opens promising perspectives for the appli-cation of SMHT in different biomedical fields. Next steps couldbe the use of SPIONs in an animal model of obesity, to analyzethe metabolic pathways activated by the SMHT, and the applica-tion of SMHT in an experimental model of cancer in obese miceto study the crosstalk between adipose and tumor tissues.

ReferencesLee JH, et al. Nat Nanotechnol 2011;6:418-22.Guardi P, et al. ACS Nano 2012;6:3080-91.Orlando T, et al. Contrast Media Mol Imaging 2016;11:139-45.Bernabucci U, et al. J Mol Endocrinol 2009;42:139-47.Sharifi S, et al. Sci Rep 2013;3:2173.Brasaemle DL. J Lipid Res 2007;48:2547-59.

7


BIODERIVED POROUS SILICA HYBRID NANOVECTORFOR THERANOSTIC APPLICATIONSR. Moretta1,2, M. Terracciano1, I. Rea1, P. Dardano1, L. DeStefano1

1Institute for Microelectronics and Microsystems; 2Department ofChemistry, University of Naples “Federico II”, Naples, Italy


Theranostic nanomedicine, which represents the development ofintegrated nanotherapeutic systems able to diagnose, deliver target-ed therapy and monitor the response to the therapy, has led to a sig-nificant evolution in the research in the biomedical field. Innovativetheranostic nanodevices could be designed by combining nanoparti-cles (NPs) with peculiar properties, able to deliver both diagnosticimaging agents and therapeutics at the same time. To this aim, sev-eral kinds of inorganic and organic nanoplatforms have been devel-oped1. Semiconductor quantum dots, magnetic and gold NPs arefew examples of inorganic NPs proposed for fluorescent, magneticand photoacoustic imaging, respectively2,3. Moreover, syntheticporous silica nanostructures were successfully used as carriers fordrug delivery, despite their safety has not been demonstrated yet4.In order to develop biocompatible nanovectors, natural porous sili-ca, obtained by diatoms, were recently exploited. Such inorganicnanoplatform is a cheap fossil compound, formed by fragments ofdiatom skeletons, called frustules5. The main constituent ofdiatomites is amorphous silica, approved as food grade by Food andDrug Administration (FDA). In vivo studies demonstrated thatdiatomite-derived NPs did not induce observable abnormalities inthe organs of the mice nor tissue damage6. Herein, we explore thefabrication of a hybrid multifunctional nanodevice made of polyeth-ylene glycol (PEG)-modified DNPs (PEG-DNPs) covered by goldNPs (AuNPs) for theranostic applications. Preliminary tests ofuptake in HeLa cells, using AuNPs/PEG-DNPs labelled with Alexa488, demonstrated the perinuclear localization of the nanocom-plexes.

References1.Lim E-K, et al. Chem. Rev 2015;115:327-94.2. Gao X, et al. Nat Biotechnol 2004;22:969-76.3. Wang S, et al. Theranostics 2016;6:2394-413.4 Park. JS, et al. Small 2011;7:2061-9.5. Rea I, et al. Biochim Biophys Acta 2014;1840:3393-403.6. Delalat B, et al. Nat Commun 2015;6:8791.

PHYSICO-CHEMICAL CHARACTERIZATION OF DRUGNANOCARRIERS: A CRUCIAL STEP OR A WASTE OFTIME? S. Sennato1,2, A. Sarra2, C. Panella La Capria2, C. Bombelli3, E. Donati3, F. Bordi2

1Institute of Complex Systems (ISC), CNR, Rome; 2Departmentof Physics, La Sapienza University of Rome; and 3Institute forBiological Systems (ISB), CNR, Rome, Italy


Recently great effort has been devoted to the development of nano-sized-carriers of different nature able to efficiently entrap biologi-cally active compounds. Notably, materials with nanometric dimen-sions have distinct physicochemical properties in terms of size, sur-face properties, shape, composition, molecular weight, purity, stabil-ity, and solubility, which are crucial determinants of their physiolog-ical behaviour1. The key parameters in the physicochemical proper-ties of a nano-formulation that are typically delineated in the liter-ature are drug entrapment, stability, release kinetics and particlesize and zeta potential. More, the so desired actual number of par-ticles in formulations is of importance for comprehensive physico-chemical characterization, pharmacodynamics and quality assur-ance. Currently there are no validated experimental methods fordetermining the particle number-concentration (Nc) of nanoparti-cle formulations. since theoretical calculation methods rely on sev-eral assumptions2 and other empirical counting methods suffer of

different limitations3. Here we propose a new method for determin-ing the number of particles based on high performance LaserTransmission Spectroscopy (LTS)4 which measures the transmit-tance of a laser beam through a suspension of particles as a func-tion of the wavelength. The particle density distribution, as a func-tion of their size, can be calculated through the Beer-Lambert lawtechnique. Compared with traditional diffusion techniques (staticand dynamic light scattering), LTS allows not only to study the sizeand shape of the particles in suspension but also to determine theirabsolute concentration Nc. In this study measurements on liposo-mal suspensions of HSPC:Chol and HSPC:DPPG prepared withextrusion techniques followed by sephadex purification have beenperformed by LTS and particle size distribution and Nc have beendetermined. Results obtained by LTS have been compared withthose obtained by dynamic light scattering to shed light on somecritical issue of this commonly used characterization technique.

References1. Manaia E, et al. Int J Nanomedicine 2017;12:4991-5011.

2. Epstein H, et al. Biomaterials 2006;27:651-9,

3. Sowerby S, et al. Sensors Actuators B 2007;123:325-30

4. Li F, et al. Appl Optics 2010;49:6602-11.

NANOMATERIALS IN THE FRAMEWORK OF REACH &CLP REGULATIONSM. Alessandrelli, R. Draisci

National Centre of Chemicals, Cosmetics and ConsumerProtection (CNSC), Istituto Superiore di Sanità (ISS), Italy


Nanomaterials and nanotechnologies are a high-powered technical-scientific field and new challenges are emerging in the legislativeframework regarding the specific evaluation and adequate manage-ment of potential risks related to nanomaterials. Currently no spe-cific legislation solely devoted to nanomaterials is available butnanomaterials are covered by existing sector-specific legislations(cosmetics, biocides, food, medical devices)1. Nanomaterials arechemicals and as such they are included in the framework ofREACH (EC Regulation No. 1907/2006)2 and CLP (ECRegulation No. 1272/2008)3, overarching regulations that apply tochemicals in any size, shape or physical state. CLP4 is based onintrinsic hazardous properties of the substances and mixtures: thenanospecific behaviour suggests that specific guidelines arerequired. CLP does not yet contain specific definitions or provisionsfor nanomaterials whereas for REACH, on 3 December 2018, theEU Commission adopted the Regulation (EU) 2018/18815 whichintroduces the nanoforms of substances and nanospecific prescrip-tions by modifying the annexes I, III, VI, VII, VIII, IX, X, XI andXII. The EU Commission, in cooperation with the EuropeanChemical Agency (ECHA) and world organizations, such as GHS-ONU and OECD, recognize the compelling need to modify existingregulations, adapting them to nanospecific perspective. Scientificdevelopments have led to promote the systematic amendmentreview of OECD test guidelines and technical documents. To makesure of the significant and safe impact of nanomaterials placed onthe market, it is important to integrate the high technical advan-tages by applying these materials with the scientific results of therisk evaluation in order to ensure regulatory oversight for nanoma-terial and safety management of nanomaterials.

References1. Mech A, et al. Nanotoxicology 2019;13:119-141

2. EU Regulation (EC) No. 1907/2006 concerning the Registration,Evaluation, Authorisation and Restriction of Chemicals (REACH).

3. EU Regulation (EC) No. 1272/2008 on clas sification, labelling andpackaging of substances and mix tures, OJ L 353

4. Alessandrelli M, Polci ML. Annali ISS 2011;47:146-52.

5. Commission Regulation (EU) 2018/1881 amending REACH as regardsAnnexes I, III,VI, VII, VIII, IX, X, XI, and XII to address nanoforms ofsubstances OJ L 308.

8


therapeutic nanoproducts: ejh

Documents