br The evidence that CCL administration

The evidence that CCL660 administration increased levels of miR-660 also in non-target organs, such as lung, liver and spleen, prompted 
us to investigate whether miRNA over-expression could have side ef-fects on normal tissues. In this regard, it is worth mentioning that though human hsa-miR-660 is not conserved in the mouse, a murine miRNA having the same seed sequence as miR-660 exists, namely mmu-miR-6987 (Fig. 4A). The two miRNAs are hence expected to share part of their targets, provided that the latter have conserved binding se-quences in their 3’UTRs. This is the case of MDM2, because murine
ortologue Mdm2 is predicted to be target of both human miR-660 and murine miR-6987 (Fig. 4A). Since miR-660 could potentially bind murine Mdm2, we analyzed Mdm2 mRNA expression in healthy tissues from mice treated with CCL660 and observed a statistical significant reduction of Mdm2 expression in lung, liver and spleen (Fig. 4B). De-spite this, CCL660 did not induce any major side effects on such organs, as evidenced by organ weight and immune cell infiltrates analysis performed after 4 weeks of CCL660 treatment in immunodeficient mice (Fig. S3A-B).
The safety of the cationic lipid DOTAP for the PTK0796 has been pre-viously demonstrated in vitro and in vivo for asODNs and siRNAs [24,25,34,35]. To confirm the evidence that normal cells well tolerate miR-660 over-expression, we analyzed proliferation rate and apoptosis in various cell types both from human and mice after miRNA trans-fection. In particular, we selected non tumorigenic human bronchial epithelial cells (HBEC-1), human kidney cells (HEK293), human hepatic stellate cells (LX-2), human endothelial cells (HUVEC) and primary human macrophages from healthy donors.
As shown in Fig. 4C we did not observe any significative changes in cell proliferation at 72 and 120 h after miR-660 transfection for all the cells analyzed. Furthermore, we evaluated apoptosis by measuring the AnnexinVpos/PIneg cells in miR-660 over-expressing cells without ob-serving changes in the number of apoptotic cells after 72 h compared to cells transfected with mimic control (Fig. 4D). MiR-660 over-expression did not induce any significative changes in proliferation and apoptosis also in mouse liver, kidney and macrophage cells (Fig. 4E-F).
Overall, these findings let speculate that though CCL660 delivers miR-660 also to non-target organs and cells, normal cells are somehow tolerant to miR-660-induced MDM2/Mdm2 down-regulation. Furthermore, our results show that MDM2 inhibition is selective and induces p53-dependent cell death only in tumor cells as already de-scribed for Nutlin and other small MDM2 inhibitors [36].
2.6. CCL660 does not induce immune-related toxic effects
It is known that liposomal compounds may have immune-related toxic effects [37]. To assess the acute in vivo toxicity of our lipidic ve-hicles we injected i.p. HEPES-buffered saline alone (CTR) or a single dose (1.5 mg/kg) of miR-660 or miR-SCR entrapped in lipidic nano-particles (CCL660 or CCLSCR) into immunocompetent mice (n = 3 animals per group) and evaluated blood biochemistry, organ weight, immune cell infiltrates in organ tissues, and chemokine, cytokine and growth factor production [38].
As shown in Fig. 5A-B,H&E staining did not reveal any signs of in-flammation in the organs, and no significant changes occurred in im-mune subpopulations in CCL660-treated mice compared with those in controls. Furthermore, no variations in the body and organ weights were observed in mice treated with lipid-nanoparticles, again con-firming that our lipidic vehicles did not exert any apparent toxic effects.
Blood biochemical analyses of metabolites (alanine transaminase (ALT), aspartate transaminase (AST), azotemia, glucose and creatinine) did not reveal significant differences between CCL660 and the control (Table 2), indicating that lipid-nanoparticles did not exert toxic effects on the liver and kidney of treated mice.