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Emanuela Cova,1 Laura Pandolfi,1 Miriam Colombo,2 Vanessa Frangipane,1 Simona Inghilleri,1 Monica Morosini,1 Simona Mrakic-Sposta,3 Sarah Moretti,3 Manuela Monti,4 Ymera Pignochino,5 Silvia Benvenuti,5 Davide Prosperi,2,6 Giulia Stella,1 Patrizia Morbini,7 Federica Meloni8 1Clinic of Lung Diseases, IRCCS Foundation Policlinico San Matteo, Pavia, Italy; 2Deparment of Biotechnology and Bioscience, University of Milano - Bicocca, Milan, Italy; 3National Council of Research, Institute of Bioimaging and Molecular Physiology, Segrate, Milan, Italy; 4Laboratory of Biotechnology, Research Center of Rigenerative Medicine, IRCCS Foundation Policlinico San Matteo, Pavia, Italy; 5Experimental Clinical Molecular Oncology, IRCCS Candiolo Cancer Institute-FPO, Candiolo, Turin, Italy; 6Laboratory of Nanomedicine, Clinical Institute of Maugeri, S.p.A., Pavia, Italy; 7Department of Molecular Medicine, Pathology Unit, IRCCS Foundation Policlinico San Matteo, Pavia, Italy; 8Department of Internal Medicine, Pneumology Unit, University of Pavia, Pavia,Italy Purpose: Malignant pleural mesothelioma (MPM) is an aggressive tumor characterized by poor prognosis. Its incidence is steadily increasing due to widespread asbestos exposure. There is still no effective therapy for MPM. Pemetrexed (Pe) is one of the few chemotherapeutic agents approved for advanced-stage disease, although the objective response to the drug is limited. The use of gold nanoparticles (GNPs) as a drug delivery system promises several advantages, including specific targeting of malignant cells, with increased intracellular drug accumulation and reduced systemic toxicity, and, in the case of MPM, direct treatment administration into the pleural space. This study aims at exploring CD146 as a potential MPM cell-specific target for engineered Pe-loaded GNPs and to assess their effectiveness in inhibiting MPM cell line growth.Methods: MPM cell lines and primary cultures obtained by pleural effusions from MPM patients were assayed for CD146 expression by flow cytometry. Internalization by MPM cell lines of fluorescent dye-marked GNPs decorated with a monoclonal anti CD146 coated GNPs (GNP-HC) was proven by confocal microscopy. The effects of anti CD146 coated GNPs loaded with Pe (GNP-HCPe) on MPM cell lines were evaluated by cell cycle (flow cytometry), viability (MTT test), clonogenic capacity (soft agar assay), ROS production (electric paramagnetic resonance), motility (wound healing assay), and apoptosis (flow cytometry).Results: GNP-HC were selectively uptaken by MPM cells within 1 hour. MPM cell lines were blocked in the S cell cycle phase in the presence of GNP-HCPe. Both cell viability and motility were significantly affected by nanoparticle treatment compared to Pe. Apoptotic rate and ROS production were significantly higher in the presence of nanoparticles. Clonogenic capacity was completely inhibited following nanoparticle internalization.Conclusion: GNP-HCPe treatment displays invitro antineoplastic action and is more effective than Pe alone in inhibiting MPM cell line malignant phenotype. The innovative use of specifically targeted GNPs opens the perspective of local intrapleural administration to avoid normal cell toxicity and enhance chemotherapy efficacy. Keywords: gold nanoparticles, nanodrug delivery, mesothelioma, pemetrexed, intrapleural delivery

The use of gold nanoparticles as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favours epithelial mesenchymal transition and activation of local mesenchymal cells leading to airway fibro-obliteration. In a previous work, we engineered gold nanoparticles loaded with the mTOR inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary mesenchymal cells isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these gold nanoparticles (GNP-HCe) were able to specifically inhibit primary mesenchymal cells without affecting bronchial epithelial cell. In the present work, we investigated the effect of the these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibro-genesis. In addition, we administered GNP-HCe by inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. Present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are nontoxic.

Aims: Chronic lung allograft dysfunction represents the main cause of death after lung transplantation, and so far there is no effective therapy. Mesenchymal cells (MCs) are primarily responsible for fibrous obliteration of small airways typical of chronic lung allograft dysfunction. Here, we engineered gold nanoparticles containing a drug in the hydrophobic section to inhibit MCs, and exposing on the outer hydrophilic surface a monoclonal antibody targeting a MC-specific marker (half-chain gold nanoparticles with everolimus). Materials & methods: Half-chain gold nanoparticles with everolimus have been synthesized and incubated with MCs to evaluate the effect on proliferation and apoptosis. Results & discussion: Drug-loaded gold nanoparticles coated with the specific antibody were able to inhibit proliferation and induce apoptosis without stimulating an inflammatory response, as assessed by in vitro experiments. Conclusion: These findings demonstrate the effectiveness of our nanoparticles in inhibiting MCs and open new perspectives for a local treatment of chronic lung allograft dysfunction. Original submitted 24 July 2013; Revised submitted 15 October 2013