1. Research Laboratory on Human Reproduction (LHR), Faculty of Medicine, Université Libre de Bruxelles (ULB), Brussels, Belgium
2. Engineering of Molecular NanoSystems (EMNS), Ecole Polytechnique de Bruxelles, Université Libre de Bruxelles (ULB), Brussels, Belgium
Abstract Text: MiRNA-based therapy is tested as an innovative pharmacological approach to prevent chemotherapy-induced ovarian damage and preserve the fertility of female cancer patients. Previously, we showed that let-7a replacement therapy using liposomes was able to prevent cyclophosphamide metabolites (4HC)-induced apoptosis in PND3 ovaries in vitro but in vivo delivery of miRNAs remains a major challenge. Gold nanoparticles (AuNPs) system appears to be attractive for their biocompatibility and their ability to be multi-functionalized, allowing the grating of the desired cargo and a future selected ligand for the recognition of a targeted tissue. The synthesis of the AuNPs is concentrated on the chemical bond that link let-7a to the surface of the AuNP. Classically, thiol (sulfur-hydrogen) was used in most of the studies that grafted miRNAs to AuNPs (AuNPs-S-Let-7a). Here, we exploited the macrocyclic molecule calix[4]arene for their robust carbon-gold bond and the possibility to add functional groups, allowing the attachment of a potential control number of miRNAs (AuNPs-X4-Let-7a) and other biomolecules. First, we showed that 3 nM of AuNPs-S-let-7a and AuNPs-X4-Let-7a were not toxic compared to the control (no AuNPs) in PND3 ovaries culture in vitro for 48h by the analysis of the expression of caspase-3 (RT-qPCR), TUNEL assay (double strand DNA breaks detection) and LDH assay (relative secretion of lactate dehydrogenase) (one way ANOVA, n ≥ 4). Then, AuNPs-X4-Let-7a were functionalized with a fluorochrome that allow the imaging of internalized AuNPs in the PND3 ovaries after 48h of transfection. After observing the safety and the ability of AuNPs-X4-Let-7a to enter the tissue, we transfected PND3 ovaries with these AuNPs 24h prior to a 24h-treatement with 4-HC (AuNPs+4-HC group) to study the protective effect of let-7a delivered by AuNPs. This condition was compared to untreated ovaries (control), ovaries treated with 4-HC for 24h and ovaries cultured with 3 nM of AuNPs-X4-Let-7a for 48h. Using the three same techniques to assess the toxicity of the 4-HC (caspase-3 expression level, TUNEL assay and LDH assay; n ≥ 4), only the TUNEL assay reported a significant higher apoptosis rate in the 4-HC group compared to control (p< 0.05, one-way ANOVA, n = 5), although a trend of a higher relative LDH secretion, released by death cells, was observed for the 4-HC group compared to the control (n = 4). Regarding the effect of let-7a to protect the ovaries from 4-HC toxicity, no significant difference was reported between the 4-HC group and the AuNPs+4-HC group, suggesting a need for improving let-7 a delivery as not optimal based on let-7 and two of its target genes expression analysis (Hmga2 and Stat3) (n = 4). To facilitate access to let-7a, 2 different strategies are currently tested (i) the addition of a spacer (10 uracils) to the passenger strand (ii) the combination of adding a spacer and introducing mismatches in the double strand of let-7a that are endogenously presented. The let-7a delivery is compared to a negative control with a non-targeting sequence. Finally, additional techniques is used to better assess the toxic effect of 4-HC and the potential protective effect of let-7a such as the analysis of other apoptotic genes expression levels and the evaluation of proteins involved in DNA repair, apoptosis and follicles activation.
