Melphalan is an efficient chemotherapeutic agent that is currently used to treat retinoblastoma (Rb); however, the inherent risk of immunogenicity and the hazardous integration of this drug in healthy cells is inevitable. MicroRNAs are short non-coding single-stranded RNAs that affect a vast range of biological processes. Previously, we focused on the regulatory role of miR-181a during cancer development and progression. In this manuscript, 171 nm switchable lipid nanoparticles (LNP) co-delivered melphalan and miR-181a with encapsulation efficiencies of 93%. Encapsulation of melphalan in LNP significantly improved its therapeutic efficiency. Gene analysis shows that miR-181a decreases the expression of anti-proliferative gene MAPK1 and anti-apoptotic gene Bcl-2, but significantly increased the expression of pro-apoptotic gene BAX. Our results suggest that the two agents have a complementary effect in reducing the viability of cultured Rb cells (primary and cell line) and decreasing Rb cell counts in an in-vivo xenograft Rb model in rats. Our results suggest that the proposed co-delivery technique significantly increases the therapeutic impact, allows for lower administration of melphalan, and consequently, could minimize the cytotoxic side-effects of this drug.

Polyethylene glycol (PEG) is used in Lipid Nanoparticles (LNPs) formulations to confer stealth properties and is traditionally anchored in membranes by a lipid moiety whose length significantly impacts the LNPs fate in vivo. C18 acyl chains are efficiently anchored in the membrane, while shorter C14 lipids are quickly desorbed and replaced by a protein corona responsible for the completely different fate of LNPs. In this context, a method to predict the biological behavior of LNPs depending on the lipid-PEG dissociation was developed using the Nanoparticle Tracking Analysis (NTA) method in serum. Two formulations of siRNA-containing LNPs were prepared including CSL3 or SM-102 lipids and were grafted with different lipids-PEG (C18, C14 lipids-PEG, and Ceramide-PEG). The impact of the lipid-PEG on the interactions between LNPs and serum components was demonstrated by monitoring the mean particle size and the concentration over time. In vitro, these formulations demonstrated low toxicity and efficient gene knockdown on tumor MDA-MB-231 cells, but serum was found to significantly impact the efficiency of C18-PEG-based LNPs, while it did not impact the efficiency of C14-PEG-based LNPs. The NTA method demonstrated the ability to discriminate between the behaviors of LNPs according to serum proteins’ interactions. CSL3 lipid and Cer-PEG were confirmed to have promise for LNP formulation.

Advanced-glycation end products (AGEs) were recently implicated in vascular calcification, through a process mediated by RAGE (receptor for AGEs). Although a correlation between AGEs levels and vascular calcification was established, there is no evidence that reducing in vivo AGEs deposition or inhibiting AGEs-RAGE signaling pathways can decrease medial calcification. We evaluated the impact of inhibiting AGEs formation by pyridoxamine or elimination of AGEs by alagebrium on diabetic medial calcification. We also evaluated if the inhibition of AGEs-RAGE signaling pathways can prevent calcification. Rats were fed a high fat diet during 2 months before receiving a low dose of streptozotocin. Then, calcification was induced with warfarin. Pyridoxamine was administered at the beginning of warfarin treatment while alagebrium was administered 3 weeks after the beginning of warfarin treatment. Results demonstrate that AGEs inhibitors prevent the time-dependent accumulation of AGEs in femoral arteries of diabetic rats. This effect was accompanied by a reduced diabetes-accelerated calcification. Ex vivo experiments showed that N-methylpyridinium, an agonist of RAGE, induced calcification of diabetic femoral arteries, a process inhibited by antioxidants and different inhibitors of signaling pathways associated to RAGE activation. The physiological importance of oxidative stress was demonstrated by the reduction of femoral artery calcification in diabetic rats treated with apocynin, an inhibitor of reactive oxygen species production. We demonstrated that AGE inhibitors prevent or limit medial calcification. We also showed that diabetes-accelerated calcification is prevented by antioxidants. Thus, inhibiting the association of AGE-RAGE or the downstream signaling reduced medial calcification in diabetes.