BIOCOMPATIBLE ADHESIVE MATERIAL, ADHESIVE SURGICAL PROSTHESIS, AND RELATIVE KIT

rICHIESTA EL BREVETTO PTC sulla base delle evidenze scientifiche documentate con i lavori scientifici pubblicati e di seguito sotto elencati: 1: Carbon nanotubes toxicology and effects on metabolism and immunological modification in vitro and in vivo M Chiaretti1, G Mazzanti2, S Bosco3, S Bellucci4, A Cucina5, F Le Foche6, G A Carru7, S Mastrangelo2, A Di Sotto2, R Masciangelo8, A M Chiaretti9, C Balasubramanian4, G De Bellis4, F Micciulla4, N Porta3, G Deriu3 and A Tiberia4 1 General Surgery Department ‘Paride Stefanini’, Rome University ‘La Sapienza’, Italy 2 Human Physiology and Pharmacology Department ‘V. Ersparmer’, Rome University ‘La Sapienza’, Italy 3 Cattedra di Anatomia ed Istologia Patologica IV, Universit`a degli Studi di Roma ‘La Sapienza’, Italy 4 INFN-Laboratori Nazionali di Frascati, Via E Fermi 40, 00044 Frascati, Italy 5 General Surgery Department ‘Pietro Valdoni’, Rome University ‘La Sapienza’, Italy 6 Divisione Malattie Infettive e Tropicali II, Universit`a degli Studi di Roma ‘La Sapienza’, Italy 7 Policlinico ‘Umberto I’, Roma ‘La Sapienza’ University Hospital, Italy 8 Medical Statistic Service, Dipartimento di Medicina Sperimentale, Rome University ‘La Sapienza’, Italy 9 Biology Sciences Faculty, Rome ‘La Sapienza’ University, Italy Journal of Physics Condensed Matter, 2008, vol. 20, 474203 (10pp) ISSN: 0953-8984, doi: 10.1088/0953-8984/20/47/474203 Abstract The aim of this research is focused on the biological effects of multi wall carbon nano tubes (MWCNTs) on three different human cell types, laboratory animals in vivo, and immunological effects. Large numbers of researchers are directly involved in the handling of nanostructured materials such as MWCNTs and nanoparticles. It is important to assess the potential health risks related to their daily exposure to carbon nanotubes. The administration of sterilized nanosamples has been performed on laboratory animals, in both acute and chronic administration, and the pathological effects on the parenchymal tissues have been investigated. We also studied the effects of MWCNTs on the proliferation of three different cell types. MCF-7 showed a significant inhibition of proliferation for all conditions studied, whereas hSMCs demonstrated a reduction of cell growth only for the highest MWCNTs concentrations after 72 h. Also, no growth modification was observed in the Caco-2 cell line. We observed that a low quantity of MWCNTs does not provoke any inflammatory reaction. However, for future medical applications, it is important to realize prosthesis based on MWCNTs, through studying the corresponding implantation effects. Moreover, it has to be emphasized that this investigation does not address, at the moment, the carcinogenicity of MWCNTs, which requires a detailed follow-up investigation on the specific topic. In view of the subsequent and more extensive use of MWCNTs, especially in applications where carbon nanotubes are injected into the human body for drug delivery, as a contrast agent carrying entities for MRI, or as the basic material of a new prosthesis generation, more extended tests and experiments are necessary. 2: Multi-Walled Carbon Nanotubes: lack of mutagenic activity in the bacterial reverse mutation assay Antonella Di Sotto§, Massimo Chiaretti†, Angela Carru†, Stefano Bellucci‡, Gabriela Mazzanti§* § Department of Human Physiology and Pharmacology, “Sapienza” University, P.le Aldo Moro 5, 00185 Rome, Italy † General Surgery Department “Paride Stefanini, “Sapienza” University, P.le Aldo Moro 5, 00185 Rome, Italy ‡ INFN Frascati National Laboratories, Via Enrico Fermi 40, 00044 Frascati (Rome), Italy Toxicology Letters, 2009, vol. 184; p. 192-197, ISSN: 0378-4274, doi:10.1016/j.toxlet.2008.11.007 Abstract The mutagenic effect of multi-walled carbon nanotubes(MWCNTs) characterised by small surface/volume ratio, high diameter and less than 0.1% of metal contaminants was evaluated by the bacterial reverse mutation assay (Ames test) on Salmonella typhimurium TA 98 and TA 100 strains, and on Escherichia coli WP2uvrA strain, in presence and in absence of the metabolic activation system S9. A preliminary cytotoxicity assay was carried out to ensure that cytotoxicity did not interfere with response. MWCNTs resulted devoid of mutagenic effect in the bacterial cellular systems tested in that they did not significantly increase the number of revertant colonies. The mutagenic activity did not even appear in presence of the metabolic activator, so we can exclude that MWCNTs metabolites, produced via cytochrome-based P450 metabolic oxidation system, may act as mutagens. Carbon nanomaterials seem to exhibit different biological activities and different toxicities in relation to their physico-chemical characteristics, size, shape, crystallinity and presence of metal traces, so it is difficult to establish their health risk. Due to the limited background of genotoxicity studies and the increased occupational and public exposure to nanomaterials, present results appear useful to extend the knowledge on the safety of carbon nanotubes in view of their possible applications. 3: Preliminary results on toxicology and mutagenic effects of buckypaper (BP) and multiwalled carbon nanotubes (MWCNTs) on metabolism and immunological modification in vitro and in vivo. M. Chiaretti1, A. Cucina2, S. Bellucci3, A. Di Sotto4, G. Mazzanti4 (1) General Surgery Department “Paride Stefanini”, Rome University “La Sapienza”, Italy (2) General Surgery Department “Pietro Valdoni”, Rome University “La Sapienza”, Italy (3) INFN-Laboratori Nazionali di Frascati, Via E. Fermi 40, 00044 Frascati, Italy (4) Human Physiology and Pharmacology Department "Vittorio Ersparmer", Rome University “La Sapienza”, Italy Abstract Book of the 20th International Congress on Anti-Cancer Treatment (ICACT), Paris 3-6 February 2009, p. 438-439 Abstract The aim of our research is focused on the biological effects of BP and functionalized MWCNTs in laboratory animals in vivo, on the immunological buckypaper effects on mice and the effects of functionalized MWCNTs on five different cell types and finally, lack of mutagenic activity in the bacterial reverse mutation assay of MWCNTs. Nanomaterials possible applications in surgery, cancer therapy and new imaging techniques involve large numbers of researchers and potential patients. It is important to assess the potential health risks related to their exposure to nanoparticles [1, 2, 3]. The implantation of sterilized BP followed the study of serum immunological modifications after MWCNTs intraperitoneal administration without antigenic humoral reactions and humoral immunity [5]. We also studied the effects of MWCNTs not functionalized on the proliferation of three different cell types. [5, 6, 7] and the effects of BP on the proliferation of five different cell types (CaCo-2 Colorectal Carcinoma cell line; MCF-7 human breast carcinoma cell line, HL-60 human promyelocytic leukaemia cell line; HASMC normal human artery smooth muscle cells and finally HF normal human fibroblasts). It seems, at a preliminary observation, that proliferation of the cancer cell lines CaCo-2, MCF-7 and HL-60 are inhibited by BP as compared to controls, whereas there is no significant modification in proliferation of the two normal primary culture cells HASMC and HF. Pathological effects on the parenchymal tissues of laboratory animals have been studied. We also studied the effects of functionalized MWCNTs on the proliferation of three different cell types: MCF-7, HASMCs, Caco-2 cell lines. We observed also that MWCNTs, evaluated in the Ames test on Salmonella typhimurium TA 98 and TA 100 and on Escherichia coli WP2uvrA, in presence and in absence of the metabolic activation system S9, resulted devoid of mutagenic effect and reduced significantly the number of spontaneous revertants in TA 98 (up to a 35.8% reduction) [4]. In view of their subsequent and more extensive use, as to say in applications where carbon nanotubes are injected into the human body for drug delivery [9] as contrast agent carrying entities for MRI [10], or as the material of a new prosthesis generation [5], further extended tests and experiments are necessary. [5] M Chiaretti, G Mazzanti, S Bosco, S Bellucci, et Al. Carbon nanotubes toxicology and effects on metabolism and immunological modification in vitro and in vivo J. Phys.: Condens. Matter 20 (2008) 10pp. 4: Buckypaper of Multi-Walled Carbon Nanotubes (MWCNTs) toxicology and effects on metabolism, in vitro and in vivo. Stefano Bellucci3, Massimo Chiaretti1, Alessandra Cucina2, Angela Carru1 1 General Surgery Department “Paride Stefanini” “La Sapienza” University, Viale del Policlinico, 155, 00161 Rome, Italy 2 General Surgery Department “Pietro Valdoni“ “La Sapienza” University, Viale del Policlinico, 155, 00161 Rome, Italy 3 INFN Frascati National Laboratories, Via Enrico Fermi 40, 00044 Frascati (Rome), Italy Massimo Chiaretti General Surgery Department “Paride Stefanini, “Sapienza” University, Viale del Policlinico, 155, 00161 Rome, Italy tel: +39 06 4997214; fax: +39 06 4441255; e-mail: massimo.chiaretti@uniroma1.it Submitted to Nanomedicine, Future Science Group, London Abstract The aim of this research is focused on the biological effects of buckypaper (BP) of MWCnts. These materials were tested on five different human cell types in vitro, and implanted in laboratory mice. We studied the biological effects and toxicology in vitro and in vivo. Large numbers of researchers are directly involved in the handling of nanostructured materials such as MWCnts and nanoparticles. It is important to assess the potential health risks related to their daily exposure to carbon nanotubes. The administration of sterilized nanosamples and BP were been prepared and performed on cells and mice. Pathological effects on mouse tissues cicatrisation have been investigated in optical microscopy. We studied tissues modifications after buckypaper implantation along five weeks after the operation. No mutagenic effects were observed. We studied BP effects on cell proliferation of five different types of human cell cultures: two solid cancer cell lines, Caco2 and MCF-7, a leukemic cell line HL-60 and two normal primary cells, HA-SMCs (human arterial smooth muscle cells) and HFs (human dermal fibroblasts). This study showed that BP induced a significant reduction of cell proliferation in Caco2 and MCF-7 cell lines, and a complete inhibition of cell growth in HL-60 cell line. This effect of BP seems to be specific in transformed cells, because BP did not produce any reduction of the proliferation and viability of two normal primary cell cultures. Present results are useful to extend the knowledge on safety of BP in view of its possible applications. However, for future medical applications, it could be useful to realize MWCnts based prosthesis to study the implantation effects. Moreover, it has to be emphasized that our research group it is going on investigation about apoptosis and carcinogenicity of MWCnts, which requires a detailed follow-up investigation on the specific topic. In view of the subsequent and more extensive use of MWCnts, especially in medical and biological applications where MWCnts are injected into the human body for drug delivery, as a contrast agent carrying entities for MRI, or as the basic material of a new prosthesis generation, more extended tests and experiments are necessary. 5: Carbon nanotubes buckypaper toxicity: in vitro and in vivo effects on the metabolism and immunological modifications Stefano Bellucci1, Massimo Chiaretti2 1 INFN Frascati National Laboratories, Via Enrico Fermi 40, 00044 Frascati (Rome), Italy 2 General Surgery Department “Paride Stefanini” “La Sapienza” University, Viale del Policlinico, 155, 00161 Rome, Italy Talk presented at the National Nanomedicine Conference NNC, 28, 29 november 2008, Centro Biotecnologie Avanzate (CBA), Genova Abstract We studied biological effects of Multi-Walled Carbon Nanotubes (shortly MWCnts) buckypaper on laboratory animals in vivo, as well as its immunological effects on mice. We also reported on the effects of functionalized Cnts on three different cell types and the lack of mutagenic activity in the bacterial reverse mutation assay of MWCnts. New uses of nanomaterials in surgery involve large numbers of researchers and potential patient. The implantation of sterilized buckypaper followed the study of serum immunological modifications after Cnts intraperitoneal administration without antigenic humoral reactions nor humoral immunity. We also studied the effects of functionalized Cnts on the proliferation of three different cell types: MCF-7, hSMCs, Caco-2 cell lines. We observed also that (MWCnts), evaluated in the Ames test on Salmonella typhimurium TA 98 and TA 100 and on Escherichia coli WP2uvrA, in presence and in absence of the metabolic activation system S9, resulted devoid of mutagenic effect and reduced significantly the number of spontaneous revertants in TA 98 (up to 35.8% of reduction). [1] M Chiaretti, G Mazzanti, S Bosco, S Bellucci, et Al. J. Phys.: Condens. Matter 20 (2008) (10pp) [2] S. Bellucci, M. Chiaretti, et al. (2008) Nanotoxicology,submitted, Preliminary study on IR active modes in sterilized carbon nanotubes: a sterilization procedure for in vivo nanotoxicity tests.

“L’effetto Geco” dei nanotubi di carbonio era già noto così come le applicazioni in ambito aerospaziale. La nostra ricerca ha documentato che l’adesività dei nanotubi di carbonio è presente anche in ambito biologico (bioadesività dei nanotubi di carbonio sia nel formato granulare che nel formato buckypaper). Tale bioadesività è importante perché potrebbe essere utile nel fissaggio dei tessuti protesici attualmente in uso (polipropilene, polietilene e PTFE nelle varie e possibili combinazioni) ai tessuti biologici viventi sia animali che umani. E’ stata dimostrata dalle nostre ricerche una innocuità sostanziale dei nanomateriali in particolare del buckypaper che applicato sulle reti protesiche, ne permette il tenace fissaggio ai tessuti senza l’impiego di altri materiali, come ad esempio le colle biologiche oppure i fili di sutura. Nel primo caso per i possibili teorici rischi connessi all’impiego di tutti i materiali biologici che potrebbero trasmettere malattie infettive non ancora conosciute e gli elevati costi delle colle biologiche. Per i fili, le suture possono determinare l’intrappolamento di terminazioni nervose che potrebbero rimanere strozzate dal nodo di sutura e provocare neuralgie durevoli di difficile trattamento e disconfort per i pazienti. VANTAGGI TECNICI: la bioadesività è importante perché è utile nel fissaggio dei tessuti protesici attualmente in uso (polipropilene, polietilene e PTFE nelle varie e possibili combinazioni) ai tessuti biologici umani perché: 1. permette di velocizzare la procedura chirurgica ad esempio di plastica di ernia inguinale, plastica di laparocele, ancoraggio della vescica ptosica, prolassi del pavimento pelvico, rotture traumatiche del diaframma, ernia ombelicale; 2. il tenace fissaggio da buckypaper ai tessuti escludendo l’impiego di fili di sutura previene il possibile intrappolamento di terminazioni nervose che potrebbero rimanere strozzate dallo stesso nodo di sutura e provocare nel paziente neuralgie durevoli, di difficile trattamento; 3. la riduzione della durata dell’intervento chirurgico permette l’estensione di tale chirurgia, anche a pazienti cardiologicamente e/o pneumologicamente compromessi che altrimenti non possono essere operati; VANTAGGI ECONOMICI: 1. abbreviando i tempi di sala operatoria si abbattono i relativi costi gestionali ed a parità di orario lavorativo si possono realizzare un maggior numero di interventi; 2. il tenace fissaggio ai tessuti senza l’impiego di colle biologiche mette al riparo il paziente, dai possibili rischi teorici connessi all’impiego di tutti i materiali biologici che potrebbero trasmettere malattie infettive non ancora conosciute e agevola il risparmio economico delle amministrazioni sanitarie abbattendo l’esborso dovuto agli elevati costi delle colle biologiche a volte impiegate; AREE DI APPLICAZIONE PRINCIPALI Chirurgia Generale, Chirurgia Vascolare, Chirurgia Colonproctologica, Chirurgia Ginecologica, Chirurgia Urologica IMPEGNO ALLA RISERVATEZZA: "I sottoscritti si impegnano a garantire assoluta riservatezza in merito all'oggetto dell'invenzione per la quale chiede tutela brevettuale, fatta salva la possibilità di pubblicare/divulgare i relativi dati in un momento successivo alla data di deposito della domanda di brevetto". Roma, 07/05/2009 FIRMA DEI RICHIEDENTI/PROPONENTI