Newsletter. Nanotechnologies: an image in the process of being defined... 36

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1 Newsletter N u m e r o 1 1 m a r z o Editoriale... 3 ricerca & sviluppo Nanomechanical transducers based on cantilevers for bioanalysis... 5 Innovative polymer - and lipid - based nanotechnologies for drug and nucleic acid delivery... 9 Interactions of engineered nanomaterials with biological systems: models and methods to assess risk determinants The research activities of DIMA on nanomaterials Nanotecnologie per il tessile e abbigliamento Le nanotecnologie e lo sviluppo di una specifica normativa tecnica NANOTECNOLOGIE & SOCIETà Nanotechnologies: an image in the process of being defined Notizie Investire in cervelli: Siglato un importante accordo tra Mapei e l Università di Padova Gruppo di Lavoro Nazionale per l individuazione di misure di prevenzione protezione connesse con l esposizione a nanomateriali in ambito lavorativo FramingNano project publishing the first deliverables ObservatoryNano project gears-up Nanochallenge 2008 premia la nano-scrittura di ThunderNIL e il super polimero di Chimatech NANOMAT project: Piedmont on screen CNR-TASC coordina la piattaforma europea Nanoscience Foundries and Fine Analysis Smart textiles: the Systex project The Dutch Nanotechnology Action Plan N&N: dalla ricerca alle applicazioni: nuovo volume ENEA Seminari & Convegni Italy-USA Bio Nano Business Forum Convegno Italo-Svedese sulle nanotecnologie e nanoscienze a Stoccolma Convegno Nanotecnologie, Ambiente e Sicurezza NNC - National Nanomedicine Conference Missione Italiana in Giappone Nanotec2009.it Conference Conference on nanostructured polymers and nanocomposites NSTI Nanotech BioInItaly 2009: The Italian Biotech Event Euronanoforum Nanoforum IEEE Nano Prossimi eventi Periodico di informazione sulle nanotecnologie marzo 2009 Supplemento a Notizie Airi n. 165 novembre-dicembre 2008 Anno XXIII Bimestrale Abbonamento annuo Soci Euro 49,00 Non soci Euro 70,00 Spedizione in abb. postale comma 20 lett. B art. 2 L n. 662 Roma/Romanina Pubblicità 45% Autorizzazione Tribunale di Roma n. 216 del 29 aprile 1986 Redazione AIRI: Roma Viale Gorizia, 25/c tel , fax info@nanotec.it -

2 nanotec2009.it Nanotechnology > Competitiveness & innovation for industrial growth Rome > march 31 : april 3 > 2009 National Research Council > Piazzale Aldo Moro 7 Nanotec2009.it, an International Conference jointly organised by AIRI/Nanotec IT, the National Research Council (CNR) and Veneto Nanotech, with the collaboration of the Italian Institute for Foreign Trade (ICE), represents the annual National event for nanotechnology. The Conference base on the success of Nanotec2008.it and it will maintain its focus on application, with extended goals, contents and aims. The most important Italian players in the field and renowned experts from abroad will be present. Themes of the Conference are: Nanotechnology Governance Health & Medical Devices (Nanomedicine) Sustainable Development (Energy, Transport, Environment) ICT, Electronics, and Security Made in Italy (advanced materials, agrifoodsconservation of cultural heritage,...) A distinctive feature of Nanotec2009.it will be the Networking Day, at which will be devoted the entire April the 3rd The day is organised around a number of presentations and one-to-one meetings aimed to promote research and industrial collaborations, which will see the presence also of representatives from Industry, Research Institutions, and Venture Capitalists coming from China, Japan, USA and Italy. info@nanotec2009.it Organizers In collaboration with Italian Institute for Foreign Trade

3 p r i m o p i a n o t Editoriale Sono ormai cinque anni da quando, alla fine del 2003, AIRI/ Nanotec IT ha preso avvio ed è tempo di fare un primo bilancio della sua attività. Come indicato nel suo atto costitutivo, la missione di AIRI/Nanotec IT è quella di contribuire a promuovere le nanotecnologie e le loro applicazioni in Italia proponendosi come punto di incontro tra mondo della ricerca, l industria e gli organismi di governo, con l obiettivo di favorire contatti e collaborazioni e fornire un quadro di riferimento e indicazioni, utili per indirizzare strategie di ricerca, scelte operative e interventi di sostegno. Seguendo questa ottica, l attività di Nanotec IT si è sviluppata in questi anni su molti fronti. Tra le prime iniziative intraprese, vi è stata, nel 2004, la realizzazione del 1 Censimento delle Nanotecnologie in Italia al quale ha fatto seguito, nel 2006, il 2 Censimento, che è stato poi integrato nel I due censimenti hanno messo in evidenza un impegno in questo campo crescente nel Paese (si è passati dalle 120 strutture censite nel 2004 alle circa 200 nel 2007), e come il settore industriale fosse il maggiore responsabile di questo incremento. La tendenza è senz altro continuata e nel 2009 è previsto un muovo censimento per fornire un quadro ancora più completo ed aggiornato della situazione. Numerose sono state le iniziative promosse per favorire la diffusione delle informazioni ed il networking. La Newsletter ed il sito web ( sono due strumenti di questo impegno, ma Nanotec IT organizza ogni anno, da solo o con altri, convegni o workshops dedicati alle nanotecnologie, che affrontano un ampio spettro di temi. Dall applicazione di queste tecnologie in settori specifici, quali la cura della salute o il tessile (dal 2005 Airi/Nanotec IT organizza a Milano un convegno su questo ultimo tema insieme a TexClubTec), alla nanometrologia (nel 2005 e nel 2007 sono stati organizzati due workshops a Torino, con l Istituto Italiano di Metrologia), alla governance delle nanotecnologie. Nel 2008, dal 10 al 13 marzo, insieme a Veneto Nanotech, è stato organizzato a Venezia un Convegno Internazionale (Nanotec2008.it), il cui programma faceva riferimento ai temi guida del progetto del Governo Industria 2015 (Risparmio Energetico; Mobilità Sostenibile; Nuove Tecnologie per la Vita; Made in Italy; Salvaguardia dei Beni Culturali), con lo scopo di mostrare come le nanotecnologie possano dare un contributo importante al raggiungimento degli obiettivi del Progetto. Forte del successo ottenuto, il convegno, questa volta con la collaborazione anche del CNR, sarà ripetuto nella primavera 2009 a Roma con obiettivi e finalità analoghe (Nanotec2009.it). L intenzione è quella fare dell evento un appuntamento annuale fisso, di riferimento per l attività nelle nanotecnologie nel Paese. Per favorire interscambi e collaborazioni in questo campo tra gli operatori Italiani e quelli di altri paesi, AIRI/Nanotec IT ha collaborato con l Istituto per il Commercio con l Estero (ICE) ed il Ministero degli Affari Esteri (MAE) per organizzare iniziative indirizzate a tale scopo in occasione di convegni o di eventi promossi ad hoc. I Paesi nei quali queste iniziative hanno avuto luogo sono stati USA, Israele, India, Russia. AIRI/Nanotec IT, oltre che con una partecipazione diretta, ha dato il suo supporto alle costituzione della delegazione Italiana, alla preparazione di materiale informativo da distribuire, alla definizione del programma e dell agenda degli incontri. I risultati sono stati positivi e la collaborazione è previsto che continui in futuro. L impegno del Centro a livello internazionale si è esplicitato anche in altre forme. AIRI/Nanotec IT, infatti, ha stabilito ed intrattiene contatti e collaborazioni con organizzazioni analoghe e strutture di ricerca di altri paesi, Europei e non, ed ha partecipato e partecipa a diversi progetti Europei di ricerca sulle nanotecnologie. Nel 6 Programma Quadro (PQ6), AIRI/Nanotec IT è stato il coordinatore di due progetti: NanoRoadMap, il cui obiettivo era quello di definire il percorso di sviluppo delle nanotecnologie al 2015 nei settori dei materiali, energia, e medicina, e NAoMITEC, 3

4 tp r i m o p i a n o volto a favorire l impegno delle PMI nelle micro e nanotecnologie. Nel 7 (PQ7) partecipa, come workpackage leader, al progetto ObservatoryNANO ed è coordinatore del progetto FramingNano, che si propone l elaborazione di un Governance Plan per lo sviluppo responsabile delle nanotecnologie. Entrambi hanno preso avvio nella primavera 2008 e termineranno, ObservatoryNANO nel 2012 e FramingNano nel Lo sviluppo responsabile delle nanotecnologie è un obiettivo prioritario dell azione di AIRI/ Nanotec IT che, in ragione di questo impegno, oltre a partecipare ai progetti suddetti, partecipa al gruppo di lavoro sulle nanotecnologie istituito da UNI, dedicato alle problematiche di standardizzazione in questo campo, a quello sui nanomateriali di ISPESL, impegnato sul versante della valutazione dei rischi potenziali associati a questi inoltre il centro intrattiene contatti con organismi e istituzioni internazionali attenti a questi temi come il Woodrow Wilson International Center for Scholars (USA) o l International Risk Governance Council irgc (CH). AIRI/Nanotec IT è insomma ormai una realtà consolidata nel panorama delle nanotecnologie del nostro Paese, che opera in sintonia con e collega quanti, nell ambito della ricerca pubblica ed in quello delle imprese, sono attualmente impegnati in questo cam- po. La gran parte di questi sono iscritti a AIRI/Nanotec IT ed i loro rappresentanti presenti nel Consiglio Direttivo contribuiscono a indirizzarne le scelte e le azioni. La maggior parte degli obiettivi fissati al momento della sua creazione sono stati raggiunti, peroòquello di contribuire a far attivare in Italia una iniziativa nazionale per sostenere e promuovere lo sviluppo delle nanotecnologie, non è stato ancora realizzato. Una iniziativa di questo genere esiste in molti paesi, a cominciare dagli USA, ma anche in Germania, Spagna, Cina, Russia e, perfino Iran. AIRI/Nanotec IT è convinto che anche l Italia debba dotarsi di uno strumento di questo genere. Questo è anche l auspicio di quanti operano in questo settore ed anche per farsi portavoce di questa istanza AIRI/Nanotec seguiterà ad impegnarsi in questa direzione. In fin dei conti una iniziativa specifica volta a promuovere e sostenere lo sviluppo delle nanotecnologie non sarebbe altro che la razionalizzazione di un impegno che già esiste. L attivarla sarebbe però anche una scelta di tipo strategico, visto il ruolo che le nanotecnologie possono avere nella evoluzione dello sviluppo tecnologico futuro, in quanto tale iniziativa potrebbe consentire di incrementare il sostegno economico alle attività di R&S e, soprattutto, consentirebbe di indirizzare meglio gli sforzi e di ottimizzare l uso delle risorse. Elvio Mantovani Direttore AIRI/Nanotec IT 4

5 R I C E R C A & S V I L U P P O t Nanomechanical transducers based on cantilevers for bioanalysis Roberto Raiteri*, Suman Cherian*(1), Paolo Bonanno*, Alessandro Garibbo** * Department of Biophysical and Electronic Engineering, University of Genova, Via dell Opera Pia 11a, Genova, Italy (1) Currently at ST Microelectronics, Singapore ** SelexCommunications, via Pieragostini 80, Genova, Italy Introduction The development and the diffusion of scanning probe microscopes (the scanning tunneling microscope (STM) and the atomic force microscope (AFM) in particular) has boosted the research in nanotechnology in the last twenty years, not only by providing new imaging techniques with atomic resolution, but also by offering the possibility to probe directly molecular interactions and to manipulate matter with nanometer precision. These capabilities are enabled mainly by the peculiar properties of the AFM probe: an extremely sharp tip integrated at the free end of a highly flexible, microscopic cantilever structure. A cantilever is a long, thin beam, fixed at one end and free to move at the opposite one. Commercially available silicon microcantilevers are typically few hundreds (up to 700) of micrometers in length, tens of micrometer in width and one (or even less) micrometer in thickness. They are characterized by a purely elastic behaviour (i.e. they follow Hooke law) when deformed with a very low spring constant (down to few mn/m) and a high resonance frequency (in the order of tens of KHz in air), which results in dramatically limiting oscillations that may get caused by external mechanical noise. It is therefore possible to detect changes in the deflection of few nanometers without the need of antivibrating systems. In the last decade, transducers based on structures similar to AFM cantilevers have attracted an increasing interest throughout the international research community 1-6. This interest was and is mainly due to the merging of different originally independent technologies and know-hows, such as silicon microfabrication techniques and surface functionalization biochemistry, together with the development of multi-cantilever detection methods, thus offering new opportunities in physical and (bio)chemical sensing. Indeed, microcantilevers can transduce a huge number of different kinds of signal, e.g. mass, temperature, heat, electromagnetic field and stress into a mechanical deformation that can be noticed and measured either through measuring a bending or a change in the resonance frequency, with a resolution which is orders of magnitude higher than that achievable with macroscopic structures. Fig. 1 summarizes these transduction principles. Coating the cantilever with a bioreceptor, which can range from single molecules such as nucleic acids or proteins up to whole cells, results into a biosensor capable to detect and quantify the presence of a biological and/or chemical target species with high selectivity and sensitivity. In the remaining of this paper, we will describe the work done developing a new sensing platform based on an array of microcantilever for bioanalytical purposes. Key characteristics will be described: the technique implemented to measure changes in the deflection of the microcantilevers belonging to the array and the design of the fluidic system, necessary to convey the sample to the sensing area, i.e. to the functionalized free end. We then focus on just one kind of bioanalysis: DNA sequence detection via hybridizationa of single stranded oligonucleotides and melting of double stranded oligonucloetides. The immobilization strategy implemented to immobilize the probes onto the cantilever surface and some experimental results should give a glimpse of the potential of the technique, and of the issues still open as well. Cantilever arrays and deflection detection The array we used in the experiments is composed of sixteen identical rectangular silicon cantilevers and four reference mirrors grouped into four separate wells as shown in Fig.2a. The reference mirror can be used to measure the absolute cantilever bending by calculating the difference in the output signals between the cantilever and the reference mirror on the position sensitive detector (PSD) as shown in Fig.2b. Cantilevers are fabricated from single crystal silicon and are 500 µm long, 150 µm wide, and 1 µm thick (Silex Microsystems, Bruttovagen, Sweden, Fig.2c). The upper side of each cantilever is coated with a 5 nm Ti/W adhesion layer and a 30 nm Au layer. The cantilevers in each well have a 5

6 tr I C E R C A & S V I L U P P O pitch of 250 µm, respectively. The supporting chip is 22 mm long, 5 mm wide and 500 µm thick. Optical readout MCS deflections are monitored using an optical beam deflection readout which employs a linear array of vertical cavity surface emitting lasers (VCSELs, wavelength of 760 nm) and an array of microfocusing lenses. The laser spots focused on the cantilever free ends are reflected by a gold surface to a linear PSD. The laser power can be adjusted individually for each VCSEL to obtain a desired intensity signal at the PSD. The PSD (20x20 mm2 sensing area) position can be adjusted with two micrometer screws to align reflected laser spots. With this design, cantilever bending can be measured down to 0.1 nm. The interference on the PSD from the external light can be minimized by housing the whole setup in a closed chamber. Flow cell and fluidic delivery system To deliver the carrier fluid and different sample solutions to the array, a syringe pump fluidic system was used. Each well is connected to one syringe, which can then be operated in parallel. The system consists of four valves for the carrier fluid, four syringes, four sample loops, four injection valves for sample solution, and one cartridge containing the fluid cell with the four wells (Fig.3). When the cantilever array is mounted in the cell, each well is isolated by an individual Teflon gasket. This enables the introduction of different carrier fluids and sample solutions into the different wells, thus allowing in parallel analysis of various recognition assays. The whole cartridge containing the flow-through cell can be heated up to 70 C using a resistor embedded in the metal body of the cartridge itself, together with two temperature sensors. Syringes are driven by a single stepper motor and can dispense carrier fluid to the four wells simultaneously and at the same rate in the range µl /min. Between each syringe and the corresponding well, there is a standard sample injection loop connected to a six-port injection valve which is used to interchange the flow stream. Results Monitoring DNA hybridization and determination of melting temperature The detection of specific DNA sequences from biological sample is fundamental in different application fields, such as diagnostics, food analysis and detection of bacteria and viruses in the environment. Most of DNA detection techniques rely on the highly specific hybridization interaction between complementary sequences, i.e. a known sequence (also called probe) is allowed to interact with an unknown sequence (also called target); if hybridization takes place, one knows that the probe and target share a complementary nucleotide sequence 7 8, 9. We therefore immobilized short DNA sequences (25 bases) onto gold coated cantilevers using thiol-on-gold chemistry. (fig.4), and studied the cantilever deflection response when introducing the target DNA sequence in the solution. The diagram in fig.5 shows ten signals from the same array: the deflection of four cantilevers coated with a certain sequence (probe1, red curves in the diagram), the deflection of four cantilevers coated with a scrambled sequence (i.e. the same number of oligonucleotides as probea, but in a different, random sequence: probeb, blue curves in the diagram) and, as a reference, the signal from two reference mirrors (green curves). After having flowed a saline buffer solution for several hours in order to let the system to reach equilibrium, we introduced at t = 9 min, a target sequence complementary to probea. All cantilever coated with probe1 did respond by bending 25 nm from their starting position. On the other hand, the cantilevers coated with probeb bent only few nanometers. By flowing the buffer solution back, the cantilevers do not return to their original positions, thus showing a non reversible adsorption, as expected. Another parameter, which can provide information on the nucleotide content and about the sequence of double-stranded DNA molecules is the temperature at which the two strands of DNA separate in solution (DNA melting temperature) We therefore used cantilevers where hybridization was detected and, consequently, double strands of DNA were formed, and compared their deflection response to temperature changes with cantilevers coated with single-stranded oligonucleotides only. Fig. 6 shows the deflection of these cantilevers while the temperature was increased at 1 o C steps. Since the cantilevers are coated with gold on a side, they are sensitive to temperature (the well-known bimetallic effect), i.e. they bend away from the gold coated side when temperature increases, as it is clearly shown in the diagram. Ideally, without considering the transients due to thermal stabilization, the deflection varies linearly with the temperature. There is a certain temperature value, however, where the behaviour of the cantilever coated with the double-stranded DNA (red curves) differs from the cantilever coated with one strand only (blue curves), this can be clearly seen in the inset of fig.6. At T = Kelvin the red curves show a local minimum, which correspond to a transient small bending (few nanometers) in the opposite direction (toward the gold side) of the cantilevers. This phenomenon can be explained as a bimetallic response to a local decrease in the temperature due to phase transition (DNA melting) that is taking place, precisely at that specific temperature, on the cantilever surface. As a matter of fact, on the cantilevers with only single-stranded DNA, this behaviour was not observed. Conclusions In this report we described a possible sensing platform based on the bending of an array of microcantilevers together with some 6

7 R I C E R C A & S V I L U P P O t experimental results showing the capability to real-time monitoring, without the need of any label and getting high sensitivity, a fundamental biochemical process such as DNA hybridization and melting. Microcantilever sensors present several unique advantages: as shown, they can be applied to perform direct, label free detection and, at least in principle, they feature extremely high sensitivities due to their micro- nano-scopic dimensions; moreover, because of their small size, it is possible to develop large arrays of sensors and only very limited quantities of analyte are needed for detection. A small volume also means fast kinetics and short analysis time. Using batch silicon microfabrication technique to build microcantilever-based arrays allows mass production, reproducibility, and makes it possible to integrate these arrays with on-chip fluidics and electronics (e.g. for detection, data processing and transmission).however, the most promising feature probably resides in the flexibility of the transduction principle: in fact, microcantilever-based arrays can work in vacuum, gaseous, and liquid environment. The same simple mechanical structure can transduce force, mass, viscosity, heat and stress into a bending or a change of its resonance properties (frequency and Q factor). All the aforementioned features make micro- and nano-cantilevers a flexible general tool for lab-on-a-chip development. On the other hand, cantilever based sensors are still in a premature stage for real world application: a handful of technological challenges do remain and some fundamental issues are yet to be solved. From the technological point of view, main challenges affect: the reproducibility in the fabrication of small (nano) cantilevers; the development of sensitive integrated detection mechanisms and microfluidics to efficiently bring nanoliter volumes of sample onto the sensing area; the development of techniques for the reliable and automatic deposition of (bio)molecules on large arrays of small cantilevers. The mail fundamental scientific issue is developing a comprehensive theory to explain the mechanism, both at the molecular and microscopy level, of development of surface stress upon adsorption and ligand/receptor binding. This basic understanding is fundamental to optimize the efficiency of the transduction, in terms of the properties of the cantilever surface (e.g. roughness) and the bioreceptors immobilization strategy (e.g. use of flexible crosslinkers). Figure Captions: Fig. 2 (a) A sketch of the optical laser beam deflection readout system of the cantilever array. Four cantilevers and a fixed mirror are present within each of four wells (A, B, C, and D). The lasers beams are time multiplexed. (b) Cantilever bending is induced by the interactions of molecules with the sensing surface. z is the value of absolute bending between the reference mirror and the cantilever within the well. (c) Scanning electron microscope (SEM) micrograph of one well within the array. Fig.1 Schematic drawings (side view) of a variety of possible cantilever transducer principles: (a) force sensor with integrated tip for AFM; (b) bimetallic temperature and heat sensor; (c) mass loading sensor; (d) medium viscoelasticity sensor; (e) thermogravimetric sensor; and (f) stress sensor Fig.3 A schematic outline of the fluidic delivery system for the microcantilever array. 7

8 tr I C E R C A & S V I L U P P O Fig.4 Cartoon showing the lateral view of a cantilever coated with the thioled DNA probe. Oligonucleotides (25 bases) were conjugated with a short alkane chain ( (CH2)6, as a spacer) and a thiol group (HS-). Once introduced in solution the HS- binds strongly to gold thus forming a uniform monolayer of HS-(CH2)6- DNA. The different layers are not in scale. Fig.5 Sensor response to the introduction of the target DNA sequence: deflection vs. time diagram of four cantilevers coated with a sequence complementary to the target DNA (red lines), four cantilevers coated with a scrambled sequence (blue lines), and two reference mirrors (gree lines). Before and after target DNA injection a continuous flow of buffer solution was maintained. Contacts Alessandro Garibbo Selex Communications via Pieragostini, 80, Genova tel alessandro.garibbo@selex-comms.com Fig.6 Deflection response of the cantilever sensor to a stepwise temperature increase. Both cantilevers coated with a double-stranded DNA (red lines) and with single-stranded DNA (blue lines) responds to each temperature step increase bending away from the gold coated side (bimetallic effect), while the signal from reference mirrors is minimally affected (green lines). Inset: cantilever deflection in the temperature range [312.7 ; 316.7] where the average response to temperature of the cantilevers coated with the double-stranded DNA significantly differs from that of the cantilevers coated with single-stranded DNA. References 1. Thundat, T.; Oden, P. I.; Warmack, R. J., Microcantilever sensors. Microscale Thermophysical Engineering 1997, 1, (3), Moulin, A. M.; O Shea, S. J.; Welland, M. E., Microcantilever-based biosensors. Ultramicroscopy 2000, 82, (1-4), Ziegler, C., Cantilever based Biosensors. Analytical and Bioanalytical Chemistry 2004, 379, (7-8), Raiteri, R.; Grattarola, M.; Butt, H.-J.; Skládal, P., Micromechanical cantileverbased biosensors. Sensors and Actuators B-Chemical 2001, 79, Yan, X.; Ji, H.-F.; Thundat, T., Microcantilever (MCL) Biosensing. Current Analytical Chemistry 2006, 2, (3), N. V. Lavrik, M. J. S., P. G. Datskos, Cantilever transducer as a platform for a chemical and biological sensors. Review of scentific instruments 2004, 75, (7), Mukhopadhyay, R.; Lorentzen, M.; Kjems, J.; Besenbacher, F., Nanomechanical sensing of DNA sequences using piezoresistive cantilevers. Langmuir 2005, 21, (18), McKendry, I.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Guntherodt, H. J.; Gerber, C., Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array. Proceedings of the National Academy of Science 2002, 99, (15), Wu, G.; Ji, H.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M.; Chakraborty, A.; Majumdar, A., Origin of nanomechanical cantilever motion generated from biomolecular interactions. Proceedings of the National Academy of Sciences of the United States of America 2001, 98, (4), Ririe, K. M.; Rasmussen, R. P.; Wittwer, C. T., Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction. Analytical Biochemistry 1997, 245, (2), Zhou, L.; Wang, L.; Palais, R.; Pryor, R.; Wittwer, C., High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clinical Chemestry 2005, 51, (10), Reed, G.; Kent, J.; Wittwer, C., High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 2007, 8, (6), 11. 8

9 R I C E R C A & S V I L U P P O The improvement of the drug delivery and targeting, by above cited pharmaceutical technologies, received excellent impetus by the use of synthetic polymeric materials, for synthetic polymers have some striking advantages as materials for DDS. In fact polymeric materials are characterized by an excellent chemical versatility allowing the production of systems with different chemical and physicochemical properties, able to satisfy different pharmaceutical requests; in other terms, synthetic polymers can be prot Innovative polymer - and lipid - based nanotechnologies for drug and nucleic acid delivery G. Cavallaro, E. F. Craparo, G. Giammona, M. Licciardi, F. S. Palumbo, G. Pitarresi(*) Laboratory of Biocompatible Polymers Dipartimento di Chimica e Tecnologie Farmaceutiche, via Archirafi 32, 90123, Palermo, Italy (*) authors are inserted alphabetically Introduction In the recent years the research in pharmaceutical field focused its attention always less frequently on the discovery of new chemical entities, a long and expensive process, and always more often on the development of innovative technological formulations and systems (Drug Delivery Systems, DDS). These systems help drug delivery optimization, bioavailability and in general pharmaceutical performance of already known drug molecules. DDS are able to improve drug water solubility, and to increase its chemical stability, allowing its administration also through routes, otherwise not feasible. Moreover, targeted DDS are able to promote drug accumulation into the target organs, decreasing administered dose and consequently side and toxic effects. Specific advantages are offered by colloidal delivery systems, in other term nanotechnologies for delivery of bioactive agents [1]. Nanotechnologies used for modified and targeted drugs, including conventional organic molecules, peptides, proteins, nucleic acid based drug molecules (NABDs), such as sirna, antisense, are constituted by nanostructured materials at colloidal size (1-500 nm), able to release biologically active agents, chemically or physically incorporated, into specific sites and within well defined interval times, also in consequence of answer to specific stimuli: ph variation values, electric field, presence of enzymes. These systems are characterized by: nanoscaled dimensions, able to allow their direct interaction with cell components of the ill tissue at molecular level; ability to incorporate high amount of active molecules (high drug loading amounts) with subsequent increase of the efficiency of the drug delivery systems; well defined and narrow size distribution, that allows to obtain standardized drug release amount and rate; ability to delivery an intact form of chemically or physically unstable drugs, by increasing their bioavailability and decreasing administered doses, or making possible their administration, such as for nucleic acid based drugs (NABDs); ability to obtain an efficient localization of the drug in the target site, through the use of targeting portions, properly anchored on the surface of the nanosystems and characterized by high cell specificity. The preparation of efficient nanotechnologies for drug delivery needs [2]: - the proper choice of innovative materials (often based on natural or synthetic polymeric structure), depending on their compatibility, chemical and structural versatility; - biophysical and technological characterization of obtained nanosystems, particularly concerning size distribution, surface properties, porosity, changeability, ability of drug loading, physical stability, ability to constitute stable composite medicines (in the presence of proper excipients); - the proper choice of drug molecules to use particularly concerning NABDs and peptides. The great interest toward these nanostructured systems, as driving strategy in drug delivery, derives from the wide versatility of this kind of approach, that can be successfully used for the treatment of pathologies characterized by a strong impact on public health, such as tumoral pathologies, exploiting the physiopatological properties of nanosystems, of accumulating into the tumor mass by enhanced permeability and retention (EPR) and neurodegenerative pathologies (Alzhaimer, Parkinson), for the ability of some of these systems, if properly targeted, to reach CNS and here release drug molecules. 9

10 tr I C E R C A & S V I L U P P O perly tailored for specific aims for what they are designed. Among polymeric materials, polyaminoacids are a very interesting class because of their protein-like nature and the possibility of preparation by synthesis. They can be considered as a compromise between natural and synthetic macromolecules being biocompatible and with the advantage to possess a more regular arrangement and a smaller diversity of amino acid residues than natural proteins. Two very interesting synthetic polyaminoacids employed for the preparation of oral DDS are α,βpoly(n-2-hydroxyethyl)-dl-aspartamide (PHEA) [3,4] and α,β-polyaspartylhydrazide (PAHy)[5-6], both derivated from a polysuccinimide (PSI) with a high molecular weight, by reaction with ethanolamine or hydrazine, respectively. These polymers, initially proposed as plasma expanders, are water-soluble, non-toxic and non antigenic. Moveover they are produced by simple and reproducible reactions, with high yield and low cost. Among natural macromolecules useful for preparation of colloidal DDS, polysaccharides, such as dextran, hyaluronic acid and inulin, play an important role, because they are easily found, at low cost and, if necessary, susceptible to chemical derivatization to modify their physicochemical properties. In many cases, in order to modulate various intrinsic properties of oral drug delivery systems, such as for example water affinity and enzymatic biodegradability in the gastrointestinal tract, it is possible to prepare easily and at low cost, composite materials based on combination between polysaccarides and polyaminoacids, bearing or not polyethers, like PEG or polyesters like polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA). These composite materials generally show different degradation and release profiles, respect to polysaccharides or polyaminoacids alone, that allow the formulator to modulate, as he needs, these parameters thus obtaining optimized performances of dosage form. Nanotechnologies in the Laboratory of Biocompatible Polymers (Palermo University) Since many years, researchers of the Laboratory of Biocompatible Polymers of the University of Palermo work on the design, preparation and chemical and pharmaceutical characterization of new polymer and lipid based technological systems for the modified and targeted drug release. Nanostructured systems investigated in this laboratory include main nanomedicine systems, such as: macromolecular prodrugs (drug-polymer conjugates) polymeric micelles interaction complexes (polymer/protein complex) polyplexes (polymer-dna complexes) nanoparticles. Figure 1: Nanostructured systems for drug and gene delivery Macromolecular prodrugs In macromolecular conjugates, active agents are covalently linked to hydrophilic polymeric carrier by hydrolizable bonds, so that the hydrolysis of active agent/polymer linkage is necessary to its release and to obtain the effect. The main advantage of these systems is constituted by their ability to efficiently protect linked drug molecules, whereas one drawback is represented by the limited capacity to carrier drug, without undesirable changes of physic-chemical properties of drug-polymer conjugates. Different drug-polymer conjugates using PHEA as starting carrier were synthesized and characterized [7-10]; the ability of these systems to increase water-solubility and chemical stability of drugs as well as to increase bioavailabity and target accumulation was shown. [7-10]. Polymeric micelles are colloidal systems obtained by self-assembling of amphiphilic copolymers above critical aggregation concentration (CAC) through the occurring of physical interactions and in that the drug can be incorporated by physical or chemical linking to polymeric surfactant. The ability of micelle constituting polymeric surfactants to promote drug absorption increases drug membrane crossing, thus strongly improving drug bioavailability. Moreover, also polymeric micelles, for their small size can be absorbed giving circulating nanodevices able to slowly release drug molecules. PHEA based polymeric micelles were proposed to carrier antitumoral drugs as well proteins with strong improvement in drug bioavailability in vivo [12-14]. Physical polymeric complexes are nanosystems in that synthetic copolymers are properly designed and realized in order to complex by physical interactions small molecules or macromolecules. Colloidal resulting systems are able to strongly stabilize 10

11 R I C E R C A & S V I L U P P O t and protect peptide and protein molecules, allowing their administration for different administration routes. Application fields include successful oral delivery of peptides, proteins and vaccines. Therefore these polymeric materials are able to reversibly complex peptide or protein molecules (hugging like) and to release them in the intact form after absorption in blood circulation. The ability of polyaminoacidic systems to oral administer proteins was demonstrated by in vivo studies [15]. Polyplexes Polyplexes (InterPolyElectrolyte Complexes, IPECs) are physical interaction complexes obtained by electrostatic interaction between cationic polymers (positively charged) and genetic material (such as plasmids or nucleic acid based drugs, negatively charged); these systems are able to condense genetic material (until to colloidal size) and to neutralize its negative charges, in order to make possible its introduction into cells and besides they protect DNA from nuclease degradation improving transfection efficiency. PHEA and PAHy cationic copolymers have been showed to be non toxic and able to transfect plasmid to cells in vitro[16-18] Polymeric and lipid nanoparticles Polymeric nanoparticles are drug loaded nanomatrices obtained by chemical or physical crosslinking of properly derivarized macromolecules. Drug release from nanoparticles (especially from oral route) can occur by response to external stimuli such as ph or biological events, such as the activity of specific enzymes. Stealth nanoparticles based on PHEA copolymers allowed successfully to escape reticulo-endotelial system [19]. Nanoparticle can be characterized by swellable nature and in this case the constituting nanomatrice has hydrogel properties. Hydrogels are polymeric three-dimensional networks, obtained by physical or chemical crosslinking, that in contact with an aqueous medium (e.g. in a physiological/biological environment), are able to swell by taking up from 10-20% up to thousands of times their dry weight in water. As a consequence of their water affinity, hydrogels do not cause irritant effects on tissues or organs. For this reason, hydrogels are very attractive systems especially for oral drug release because they combine a good tissue biocompatibility with the possibility to modulate in appropriate way the release rate of therapeutic agents [20, 21]. stimuli-responsive hydrogels, that exhibit large volume changes in response to small changes in external environment conditions, such as ph, temperature, electric field, light, etc. Stimuli-responsive hydrogels are also indicated as intelligent or smart materials because they have both sensor and effector functions (Figure 2). In particular, ph-responsive hydrogels are frequently used to develop drug delivery systems for oral administration; in fact, the difference of ph between the stomach and the intestine is large enough to generate a ph-dependent swelling that promotes drug delivery (Figure 3). If necessary, biodegradable hydrogels can be prepared that undergo a selective hydrolysis in the intestinal tract, e.g. by colonic enzymes (Figure 3). PHEA and PAHy based hydrogels showed good biodegradability and the possibility to obtain particulate systems with well defined and narrow size distribution [22, 23]. Figure 2 Schematic representation of Smart Hydrogels Chemical hydrogels can be obtained by easy, fast, safe and at low cost methods, such as the use of chemical crosslinking agents or by UV, gamma or microwave irradiation. Taking into consideration the dependence of swelling behaviour on the environmental conditions, these systems can be produced as: conventional hydrogels, which are usually uncharged and exhibit no significant change in swelling as a function of the external environment; Figure 3 Nanosystems for colon targeted drug delivery 11

12 tr I C E R C A & S V I L U P P O Lipid nanoparticles Besides polymeric materials, also natural lipids can be used as nanoparticles to prepare colloidal drug delivery systems. These systems, due to their nature and size, can be absorbed in the intestinal tract (by enterocytes and M cells of Peyer s patches), then they arrive to the blood circulation. It is also possible to prolong their blood circulation time by producing stealth lipid nanoparticles. This allows to prolong blood time residence of drug and its efficacy. Lipid nanoparticles are particularly useful for I.v. administration of drugs for the treatment of nervous central system diseases, since they are able to cross the blood brain barrier [24]. Conclusions Nanotechnologies represent today a very promising approach to design and obtain drug and gene delivery systems able to reduce drug dose, improve drug stability and water solubility and in general optimize pharmaceutics and cell targeting and uptake of drug delivery systems. In the Laboratory of Biocompatible Polymers of the University of Palermo different nanotechnologies have been produced for drug and gene delivery; in particular: macromolecular prodrugs, able to strongly increase bioavailability of drugs and to target them toward specific cells (7, 11); polymeric micelles able to significantly increase water solubility and again bioavailability also of protein drugs (12-14); physical polymeric complexes that are able to make possible the oral administration of protein (15); polyplexes with very high transfection efficiency for gene and oligonucleotide therapy (18) and nanoparticles based on lipid or/ polymer with excellent biocompatibility and good release profile (19, 23, 24). These nanotechnologies have been successfully proposed in nanomedicine, but also their application in diagnostics and cosmetics is attractive. References 1) Tumour-targeted nanomedicines: principles and practice, T. Lammers, W. E. Hennink and G. Storm, Br J Cancer 99 (3), (2008) ) Polymer conjugates as anticancer nanomedicines, R. Duncan, Nature Reviews cancer 6 (9), ) Water-soluble copolymers of an antiviral agent: synthesis and their interaction with a biomembrane model, G. Giammona, B. Carlisi, G. Pitarresi, G. Cavallaro and V. Turco Liveri J. Control. Release 22 (1992) ) Viscosimetric investigation of the interaction between sodium dodecylsulfate micelles and a polymer drug carrier, G. Cavallaro, G. Giammona, G. La Manna, S. Palazzo, G. Pitarresi and V. Turco Liveri, Int. J. Pharm. 90 (1993) ) A new water-soluble synthetic polymer, α,β-polyasparthy hydrazide, as potential plasma expander and drug carrier, M G. Giammona, B. Carlisi, G. Cavallaro, G. Pitarresi and S. Spampinato, J. Control. Release 29 (1994) ) Conformational analysis of α,β-poly(n-hydroxyethyl)-d,l-aspartamide (PHEA) and α,β -Polyasparthydrazide (PAHy) polymers in aqueous solution, T. Coviello, Y. Yuguchi, K. Kajiwara, G. Giammona, G. Cavallaro, F. Alhaique and A Palleschi, Polymer 39 (1998) ) Chemical stability and bioavailability of acyclovir coupled to α,β poly(nhydroxyethyl)-d,l-aspartamide, G. Giammona, G. Puglisi, G. Cavallaro, A. Spadaro and G. Pitarresi, J. Control. Release 33 (1995) ) Coupling of the antiviral agent zidovudine to polyaspartamide and in vitro drug release studies, G. Giammona, G. Cavallaro, G. Pitarresi, G. Fontana and B. Carlisi, J. Control. Release 54 (1998) ) Synthesis, physico-chemical properties and biological characterization of a paclitaxel macromolecular prodrug, G. Cavallaro, M Licciardi. G. Giammona, P. Caliceti, S. Salmaso, Eur. J. Pharm. Biopharm. 58 (2004), ) Chemical conjugation of dexametasone to a polyaspartamide and in vitro evaluation studies, G. Cavallaro, L Maniscalco, G. Giammona, C. Civile, M. G. Mazzone, V. Enea, J. Drug Del. Sci. Technol. 14 (2004), ) Folate-mediated targeting of polymeric conjugates of gemcitabine, G. Cavallaro, M. Licciardi, S. Salmaso, P. Caliceti, G. Giammona, Int. J. Pharm. 307 (2006) ) Poly(hydroxyethyl) derivatives as colloidal drug carrier systems, G. Cavallaro, M. Licciardi, G. Giammona, P. Caliceti, A. Semenzato, S. Salmaso, J Controlled Rel. 89, (2003) ) Tamoxifen-loaded polymeric micelles: preparation and in vitro biological evaluation, G. Cavallaro, L. Maniscalco, M. Licciardi, G. Giammona, Macromol. Biosc. 4 (2004), ) Supramolecular association of recombinant human growth hormone with hydrophobized polyhydroxyethylaspartamides, S. Salmaso, R. Schrepfer, G. Cavallaro, S. Bersani, F. Caboi, G. Giammona, G. Tonon and P. Caliceti, Eur. J. Pharm Biopharm, 68 (2008) ) Colloidal vectors at polyaminoacid structure for oral release of peptides and proteins and its attendant production method, M. Licciardi, G. Giammona, G. Cavallaro, G. Pitarresi, International Application number PCT/IT/2008/ ) Novel cationic copolymers of a polyasparthylhydrazide synthesis and characterization, G. Cavallaro, F. S. Palumbo, M. Licciardi, G. Giammona, Drug Delivery 12 (6) (2005) ) Synthesis and characterization of novel non toxic polyaminoacidic polycations for gene therapy, M. Licciardi, M. Campisi, G. Cavallaro, M. Cervello, A. Azzolina, G. Giammona, Biomaterials 27 (2006) ) Polyhydroxyethylaspartamide-spermine copolymers: efficient vectors for gene delivery, G. Cavallaro, S. Scirè, M. Licciardi, M. Ogris, E. Wagner, G. Giammona, J. Controlled Release, 131, 54-63, ) Pegylated nanoparticles based on a polyaspartamide. preparation, physico-chemical characterization and their intracellular uptake, E. F. Craparo, G. Cavallaro, M. L. Bondì, D. Mandracchia and G. Giammona, Biomacromolecules, 7 (2006) ) Hydrogels for biomedical applications, A. S. Hoffman, Adv. Drug. Del. Rev. 54 (2002) ) Hydrogels: from controlled release to ph-responsive drug delivery, P. Gupta, K. Vermani, S. Garg, Drug Discov. Today 7 (2002) ) Photocrosslinking of dextran and polyaspartamide derivatives: a combination suitable for colon-specific drug delivery, G. Pitarresi, M.A. Casadei, D. Mandracchia, P. Paolicelli, F.S. Palumbo, G. Giammona, J. Control. Release, 119 (2007) ) Composite nanoparticles based on hyaluronic acid chemically crosslinked with α,β-polyaspartylhydrazide, G. Pitarresi, E.F. Craparo, F.S. Palumbo, B. Carlisi, G. Giammona, Biomacromolecules 8 (2007) ) Ferulic acid-loaded lipid nanostructures as drug delivery systems for Alzheimer disease: preparation, characterization and cytotoxicity studies, M.L. Bondì, G. Montana, E.F. Craparo, P. Picone, G. Capuano, M. Di Carlo, G. Giammona., Current Nanoscience 5(1) (2009) Contacts Prof. Gennara Cavallaro, gennacav@unipa.it Prof. Giovanna Pitarresi, giopitar@unipa.it Prof. Gaetano Giammona, gaegiamm@unipa.it Dipartimento di Chimica e Tecnologie Farmaceutiche, via Archirafi 32, 90123, Palermo, ITALY. 12

13 R I C E R C A & S V I L U P P O In environmental and industrial toxicology, there are established protocols and terminology describing how to assess risk and what standard of proof must be met. Accepted and standardized tests and models have been set up and are in place to allow for an evaluation of any new chemical or material against existing benchmarks and to categorize their associated risk level. Hazard identification is the process of determining whether exposure to an agent can lead to adverse health and environmental outcomes, whereas hazard characterization defines the relationship between the dose of an agent and the occurrence of adverse effects in exposed population. Being aware of the health issues concerning engineered nanot Interactions of engineered nanomaterials with biological systems: models and methods to assess risk determinants E. Bergamaschi (1), O. Bussolati (1), L. Migliore (2), R. Colognato (2), A. Magrini (3), A. Pietroiusti (3), M. Bottini (3), L. Ghibelli 3, J.M. Legramante (3), P. Boscolo (4), I. Iavicoli (5), S. Bellucci (5), A. Bergamaschi (5), S. Bellucci (5 ) 1 Dipartimento di Clinica Medica, Nefrologia e Scienze della Prevenzione e Dipartimento di Medicina Sperimentale, Università degli Studi di Parma 2 Dipartimento di Scienze dell Uomo e dell Ambiente, Università di Pisa e NMI Unit, European Commission, DG-Joint Research Centre, Institute of Health and Consumer Protection, Ispra, (VA) 3 Dipartimento di Bio-patologia e Medicina del lavoro, Università di Tor Vergata, Roma 4 Dipartimento di Scienze Bio-mediche, Università degli Studi G. d Annunzio Chieti-Pescara 5 Istituto di Medicina del Lavoro, Università Cattolica del Sacro Cuore e INFN, Frascati, Roma 1. Introduction Nanotechnology is one of the key industries in Europe [1]. The estimated economic impact of nanoparticles in industrial, consumer, and medical products will be US$ 292 billion by 2010 and US $1 trillion by 2015 [2] The prosperity of our continent and out country will also depend on the safe and sustainable development of this emerging technology. Indeed, every new technology brings with it new risks and for nanotechnology, the potential health risks to workers and consumers are paramount. They can arise from exposure to nanomaterials (NM) either at work or through consumer products. Among the stakeholders there is an increasing awareness that these risks, if not assessed and managed properly, can prevent economic growth and deprive us of a much needed competitive edge, but more importantly could have grave potential consequences for human and environmental health [3, 4]. It is acknowledged that the risks associated with NM production and use must be considered using a precautionary approach [5] and that further research is essential to inform the management of these new and emerging risks. The need to research the potential hazards of ENP is evident from several EU-funded toxicology studies underway (e.g. NANODERM - bio-kinetics of TiO 2 nanoparticles from dermal exposure-; NANOSAFE 2 - Safe production and use of NM: Development of risk assessment and management for secure industrial production of nanoparticles; PARTICLE RISK - Risk Assessment of ENP). The last call of the Sixth EU Framework Programme (FP6) funded at least two projects to study the mechanisms of interaction between engineered nanoparticles (ENP) and the living system, e.g. the NANOSH and NANOINTERACT [6]. In the first call of the Seventh EU Framework (FP7), more projects on the risk assessment of ENP have been selected. In addition, national governments - including those of the UK, Denmark, France, Germany and Belgium - are also funding studies on the toxicology of ENP. 2. Rationale for a research project aimed at assessing nanobio-interactions By tailoring the structure at the nanoscale it is possible to engineer novel materials that have entirely new physicochemical properties as compared to bulk materials. The unusual physicochemical properties of NM are attributable to the higher surface to volume ratio associated with nanoparticles (NP) and the quantum effects that occur in the nanometre scale. However, chemical composition, surface structure (reactivity, surface groups, inorganic or organic coatings), solubility, shape and aggregation should also be considered. Although impressive from a physicochemical point of view, the novel properties of NM raise concerns about adverse effects on biological systems, since they may favour enhanced uptake and interaction with several cell types and tissues [7, 8]. 13