Background Hepatocellular carcinoma (HCC) represents the second most leading cause of cancer-related death globally and the fifth most common neoplasia. Infection with hepatitis B and C viruses, as well as alcohol consumption, obesity, and diabetes are considered significant conditions that can give origin to HCC. Five-year survival of the patients from diagnosis has been estimated between 48% and 75%, and it is strictly related to the tumor resistance against chemotherapy drugs. However, the currently available limited and inefficient therapeutic options render the curative treatment of the disease difficult to be achieved; nanomedicine could be an extremely promising curative option to overcome problems of traditional approaches. Hypothesis Near-infrared (NIR)-responsive polydopamine nanoparticles (PDA NPs), able to convert NIR radiation into heat,can be exploited to kill HCC cells in a non-invasive way. As an anti-proliferation and anti-angiogenesis chemotherapy drug, sorafenib is proposed to be loaded inside PDA NPs (SRF-PDA NPs), in order to obtain chemotherapy and photothermic treatment into a single platform. Despite being the most efficient chemotherapeutic, patients usually acquire resistance against sorafenib within 6 months of therapy. As a countermeasure, delivery of sorafenib is envisioned through surface functionalization of nanoparticles with anti-EGFR antibodies; this approach moreover improves nanoparticle uptake by HCC cells overexpressing EGFR. Aims The goal of this project is development of "smart" nanoparticles based on non-invasive photothermal and combined chemotherapeutic strategies that aim to pave the way to new therapy scenarios in HCC treatment. It can be summarized in the obtainment of nanovectors able to efficiently target HCC cells and to reduce drug resistance (at the base of therapeutic failures for long-term therapy) with a multi-tasking approach Experimental Design The first step of the project is dedicated to the synthesis and characterization of anti-EGFR antibody functionalized sorafenib-loaded PDA NPs (aEGFR-SRF-PDA NPs). Thereafter, sorafenib-dependent HCC reduction will be extensively investigated jointly to the photothermal contribution in the presence of NIR laser irradiation for reduction of drug resistance. Then, the drug targeting and anti-drug resistance efficiency of aEGFR functionalization will be evaluated. Finally, systemic effects of the aEGFR-SRF-PDA-NPs will be evaluated on diethylnitrosamine (DEN)-derived mouse models. All the effects on HCC cells will be investigated at protein expression level with proteomics. Expected Results A new and improved therapeutic approach against HCC is expected to be successfully developed at the end of the project. Together with higher efficiency in HCC treatment, dose decrement of sorafenib is expected to less than 50% of current clinical protocols; a prolonged drug efficiency duration before the occurrence of drug resistance is expected. Impact On Cancer The impact of the results on cancer research will be appreciable upon the obtainment of innovative therapeutic formulation. At short term, the results will provide extensive molecular information about a combination therapy to reduce drug resistance; at long term, realistic exploitation of the nanoparticles in future clinical applications is expected. Background Hepatocellular carcinoma (HCC) is the fifth most common neoplasia worldwide and represents the third most leading cause of cancer-related death globally [1]. The risk factors increasing the incidence of HCC are hepatitis B and hepatitis C virus infections, alfa-toxin exposure, alcoholic cirrhosis, and cigarette smoking [2]. Five-year survival from diagnosis, strictly related to the stage of disease, has been estimated between 48% and 75% [3,4]. The therapeutic strategy may change on the basis of the liver conditions and of the tumor stage [5,6]; surgical resection, liver transplantation, and local ablation can improve the survival of HCC patients at an early stage diagnosis, but no effective treatments are available for patients with advanced HCC [7]. In the recent years, however, advances achieved in knowledge of molecular mechanisms underlying the growth of HCC led to the development of new entities useful for the treatment of hypervascularized HCC tumors. In vivo and in vitro studies have demonstrated that sorafenib, an oral multi-kinase inhibitor, is able to block the tumor growth by inhibiting important components of signaling pathways (e.g., vascular endothelial growth factor receptors VEGFR-2 and VEGRF-3, and platelet-derived growth factor receptor-β PDGFR-β, c-kit and Flt-31) involved in the proliferation and in the angiogenesis of the tumor [8-10]. Sorafenib also generates hypoxia in HCC cells that leads to drug resistance via HIF-1α and NF-κB activation to survive hypoxic conditions, resulting into overexpressed epidermal growth factor receptors (EGFR) [11]. In resistant cells, the efficacy of sorafenib could be prolonged by inhibiting the EGFR, as demonstrated by using two chemical inhibitors (erlotinib or gefitinib), RNA interference directed against EGFR, and a monoclonal antibody directed against EGFR (cetuximab) [12]. Specifically, designing a combined therapeutic approach including sorafenib and aEGFR antibodies could provide a prolonged chemotherapeutic efficiency derived by the chemotherapy drug. Despite being the only systemic therapy agent capable of increasing the overall patient survival, poor solubility of sorafenib in aqueous environments strongly limits its application for local treatment [13]. All considered, new and improved sorafenib-delivery strategies through HCC are required to induce an efficient therapy in absence of significant side effects. In my project, I hypothesize that a strategy based on “smart” nanoparticles (NPs) can pave the way to new therapy scenarios (Figure 1). Nanomedicine offers several very promising possibilities to significantly improve medical diagnoses and therapies, leading to an affordable, higher life quality for everyone. Nanomedicine is paving the way to develop new nano objects and smart materials capable to increase localization of therapeutic agent, with the objective to enhance efficacy and tolerance of therapy, minimizing side effects [16-18]. As far as sorafenib concerns, a limited number of examples of delivery system can be found in the literature, including dextran/poly(dl-lactide-co-glycolide) acid (PLGA) block copolymer nanoparticles [14], porous silicon nanoparticles [15], dual polymeric-lipid nanoparticles [16], PLGA microspheres [17], and solid lipid nanoparticles [18].“Smart” nanoparticles consisting in polydopamine (PDA) are exploitable as photothermal therapy agents, being able to convert light into heat, and this strategy is generally applied in combination with other chemotherapies to improve their efficiency [19]. In this scenario, metallic nanostructures like gold NPs (AuNPs) and gold nanorods (AuNRs) are widely investigated as photothermal therapeutic agents, in particular for cancer therapy [20]. A drawback of such approaches however relies in the inorganic nature of the nanovectors: the lack of substantial biodegradability is concern of systemic toxicity in long-term applications, jointly to accumulation in the reticuloendothelial system and in other excretory organs [21]. In this proposal, I aim at obtaining a satisfactory local hyperthermia by instead using highly biocompatible and biodegradable PDA NPs that will be exploited as highly effective photoconversion agents upon NIR light excitation. PDA NPs have been already proposed in photothermal cancer therapy, and showed excellent results in terms of heat generation, comparable to that one of other traditional inorganic counterparts [22]. Research Plan Recently, as a new generation and non-invasive approach, photothermal therapy in combination with chemotherapy or radio therapy has shown promising results in terms of reducing the drug resistance. The novelty in my proposal lies in the exploitation of biocompatible and biodegradable organic NPs as photothermal agents. Owing to their biodegradable nature, PDA NPs will be easily eliminated by the organism avoiding problems of accumulation and long-term toxic effects. In addition to the exploitation of the “smart” properties of PDA NPs, a chemotherapeutic treatment is also envisioned: sorafenib will be loaded in the nanovectors (SRF-PDA NPs), as it represents a new generation of chemotherapeutic drug able to inactivate VEGFR-2, VEGRF-3, PDGFR-β, c-kit, and Flt-31, and thus to inactivate a biomolecular pathway that leads to proliferation and angiogenesis of cells. This effect will also be guaranteed by further functionalization of SRF-PDA-NPs with cetuximab, that reduce the EGFR signaling, thus hindering drug resistance against sorafenib in its long term application. In vivo experiments in diethylnitrosamine (DEN)-derived mouse models own the potential to provide significant information about the systemic effects of EGFR-SRF-PDA-NPs. Upon successful project completion, collected data will pave the way for future pre-clinical studies.The project proposes a totally innovative and multidisciplinary technique in cancer therapy: as a proof of concept, it will be focused on liver cancer, but this unique approach can be extended to a wide range of neoplasia. The project is organized into 5 work-packages (WP). The time progress (36 months) and the expected durations of the each WP depicted by the Gant diagram (Table 1). WP1 is totally dedicated to aEGFR-SRF-PDA NPs synthesis and characterization. During WP2, administration of aEGFR-SRF-PDA NPs to HepG2 cell cultures and exposure to NIR radiation of different intensities and for different amounts of time will be performed in order to optimize the treatment protocol. Thereafter, the anti-drug resistance effect provided by NIR irradiation of PDA NPs and aEGFR antibodies (cetuximab) will be extensively investigated in in vitro studies (WP3). In WP4, in vivo testing of aEGFR-SRF-PDA NPs on xenograft mouse models will be investigated to have preliminary information about efficiency of aEGFR-SRF-PDA NPs on liver cancer and to evaluate their systemic effects. A proteomic analysis of HepG2 cells will be carried out upon therapeutic treatment, envisioning gene ontology analysis, and in particular analyzing molecular pathways involving 78 kDa glucose related protein (GRP78), 14-3-3ε, and heat shock protein 90β (HSP90β): in WP5 deep information about the molecular mechanisms at the base of the combined physical and pharmacological effects of aEGFR-SRF-PDA NPs will be thus provided.