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  • br To investigate the potential toxic e ects of


    To investigate the potential toxic effects of both miRNAs and delivery agents, an in vitro approach revealed that miR-660 replacement did not induce any changes in both mouse and human normal cells. Interestingly, lipid-nanoparticle delivery of synthetic miR-660 had no immunological off-target or acute/chronic toxic effects on immunocompetent mice. Altogether, our results highlight the potential role of coated cationic lipid-nano-particles entrapping miR-660 in lung cancer treatment without inducing immune-related toxic effects.
    1. Introduction
    Lung cancer is a disease with a poor prognosis, accounting for 20% of total cancer-related deaths in Europe [1]. The majority of lung cancers are represented by the non-small cell lung cancer (NSCLC) subtype that has a 5-year survival rate of < 18% [2]. Currently, surgical resection is the most common treatment for early-stage tumors and is combined with chemotherapeutic agents for patients with advanced lung cancer; chemotherapy alone is used for patients with metastatic
    Corresponding author.
    E-mail address: orazio.fortunat[email protected] (O. Fortunato). 1 These authors contributed equally to this Iberiotoxin work.
    2 These authors contributed equally to this work. 
    disease [3]. Platinum-based treatment is commonly used in clinical practice, with a small effect on improving the survival of patients with lung cancer [4]. The discovery of activating mutations in the EGFR gene (23%) and rearrangements in the anaplastic lymphoma kinase (ALK) gene (3–7%) [5] had a relevant impact on the treatment of patients with lung cancer, based on their responsiveness to tyrosine kinase in-hibitors (TKIs), such as erlotinib, crizotinib and gefitinib [6,7]. Re-cently, pembrolizumab was approved as a first-line treatment for pa-tients with lung cancer expressing high levels of programmed cell death
    ligand 1 (PD-L1) [8]. Unfortunately, targeted therapies and pem-brolizumab represent the first-line treatments for only 30% of patients [9]. Thus, the identification of novel treatment strategies remains a critical and essential need for lung cancer management.
    The tumor suppressor gene P53 plays an important role in pre-venting cancer development and is mutated/deleted in 50% of NSCLC cases [10]. P53 activity is finely controlled and, among P53 modulators, mouse double minute 2 (MDM2) plays an important role [11]. Fur-thermore, MDM2 function is linked to P53 through an autoregulatory feedback loop under both normal and pathological conditions [12,13]. Based on these considerations, strategies that restore P53 function by inhibiting MDM2 represent a potential therapy for P53 wild-type lung cancers [14].
    MicroRNAs (miRNAs) are small non-coding, 22 nucleotides long RNAs that bind complementary sequences of target mRNAs to induce their degradation or repress translation [15]. Recent studies have re-ported altered expression patterns of miRNAs in several human ma-lignancies, including lung cancer [16]. The revelation that miRNAs function as potential oncogenes and tumor suppressors has generated great interest in using them as targets for cancer therapy [17]. We previously identified miR-660 as a tumor suppressor miRNA in lung cancer cells. The replacement of this miRNA inhibits the migration and invasion of tumor cells and blocks tumor growth both in vitro and in vivo. The anti-tumor effects are mediated by MDM2 downregulation and activation of the P53 pathway [18].
    The Iberiotoxin roles of miRNAs as novel therapeutic agents have received in-creasing attention [19]. Although several pre-clinical studies examining miRNAs have been conducted over the years, only a few have transi-tioned into clinical development. Several obstacles must be overcome to envisage the clinical use of miRNA therapy. These challenges include the design of optimal miRNA delivery vehicles with higher stability or reduction of off-target effects and toxicity [20].
    Several delivery methods, such as nanoparticles, viral vectors and liposomes, are available to restore the expression of tumor suppressor miRNAs [21]. Due to their size and hydrophobic and hydrophilic characters (in addition to biocompatibility), liposomes represent a promising system for miRNA delivery because they protect and deliver nucleic acids and exhibit reduced toxicity and an improved therapeutic index [22,23]. Because Coated Cationic Lipid-nanoparticles (CCL) car-riers have been shown to efficiently deliver asODNs/siRNAs/miRNAs [24–27], in the present study, we generated novel lipidic nanoparticles entrapping miRNA-660 (CCL660) as a lung cancer treatment to explore its potential for inhibiting lung cancer cell growth. r>