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  • br Despite the encouraging results regarding


    Despite the encouraging results regarding toxicity, CCL660 only partially decreased tumor growth in P53 wild-type PDXs, and miRNA ISH revealed that CCL660 only reached 30% of lung cancer cells, thus lacking a uniform distribution in the tumor samples. This low delivery efficiency, which is likely related to rapid clearance or phagocytosis of the liposomal compound by immune cells [37], could explain the lim-ited inhibitory effects on tumor growth observed in our PDX models.
    Lipid-nanoparticles represent an effective drug delivery system that is capable of altering the pharmacokinetic profile of a drug and deli-vering the encapsulated agent [46]. One potential strategy to improve the therapeutic efficacy of the miRNA mimics and to reduce non target toxicity is to modify lipid-nanoparticles by adding a tumor cell-specific ligand on the lipid surface.
    Indeed, the combination of the pharmacokinetic advantages and tumor-selective biodistribution of lipid-nanoparticles with cell-specific binding and internalization induced by Kainic acid or receptor ligands is a recognized strategy to improve the therapeutic effectiveness of con-ventional chemotherapeutics or gene therapeutics for treating human malignancies, including lung cancer [26,47,48].
    The availability of ligands or peptides or tumor-associated antigens would enable researchers to design more sophisticated cancer treat-ment strategies that exhibit high levels of selective toxicity for cancer cells [49].
    Therefore, new candidate lung cancer cell-specific ligands must be identified to improve the therapeutic outcomes, decrease side effects and improve patients' quality of life.
    Systemic delivery is the most feasible route for the use of miRNAs in the clinic, but the main challenge is the low uptake of the delivered miRNAs in lung cancer cells [50]. The development of new delivery systems, such as inhaled liposomes, may be a valid option to overcome this issue, particularly for lung cancer treatment [51]. The success of this method will depend on the development of new aerosol devices and 
    the formulation of efficient inhalable liposomes.
    Recently, the discovery that exosomes are secreted into body fluids and their ability to be loaded with miRNAs offered a new and inter-esting approach for miRNA delivery [52]. In addition, these micro-vesicles allow the cargo to escape from phagocytosis and are endowed with an increased half-life in the circulation compared to liposomes [53]. Thus, exosomes loaded with selected miRNAs represents an al-ternative and potentially valid therapeutic strategy for treating lung cancer.
    Although challenges regarding efficient miRNA delivery persist, our experiments with lipid-nanoparticles entrapping miR-660 in preclinical mouse models showed promising results both in terms of tumor growth inhibition and the absence of toxic effects. However, future studies in pre-clinical models comparing the treatment with cationic lipid-nano-particles carrying miR-660 with the standard of care for lung cancer patients or adding a tumor cell-specific ligand on the lipid surface to improve the therapeutic efficacy of the miRNA mimic and to reduce non target toxicity are needed. Overall, the present study provides new insights into the development of miRNA delivery systems as treatments for lung cancer.
    4. Materials and methods
    4.1. Reagents and chemicals
    Hydrogenated soy phosphatidylcholine (HSPC), cholesterol (CHE), 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy (poly-ethylene glycol)-2000] (DSPE-PEG2000), and 1,2-dioleoy-1-3- ri-methylamonium propane (DOTAP) were purchased form Avanti Polar Lipids, Inc. (Alabaster, AL, USA). miRVana™ miRNA Mimic Negative Control #1 (miR-NC1) and miRVana™ miRNA mimic miR-660 (miRNA ID# MC11216) for lipid-nanoparticles preparations were purchased from Ambion (Thermo Fisher Scientific, Waltham, MA, USA).
    Nucleopore polycarbonate membranes were purchased from Avestin Inc. (Ottawa, ON, Canada). Kainic acid Sephadex G-50 was purchased from PerkinElmer Biosciences (Waltham, MA, USA).
    All other reagents were of analytical grade or the highest available purity and were purchased from Sigma-Aldrich (St. Louis, MO, USA).
    Fig. 6. MiR-660 inhibits cancer growth in lung co-lonization models. A) PET analysis of mice 28 days after the injection of 1 × 106 H460 lung cancer cells. Upper panels show representative axial sections of 18F-FDG uptake in the lungs of mice injected with H460 (left panel) and H460-miR-660 cells (middle and right panels). H and L indicate the mouse heart and lung, respectively. Lower panels show the re-sults of the ex vivo PET analysis of lungs from mice injected with H460-miR-CTR (left panel) and H460-miR-660 cells (middle and right panels). B) Representative images of H&E and pan-cytokeratin staining of the lungs from H460-miR-660-injected mice and control mice. The results revealed an in-verse correlation between miR-660 and MDM2 levels in the lungs (n = 2 mice per group).