Purpose: Cancer is a leading cause of death worldwide. According to the WHO, 8.8 million deaths were due to cancer in 2015. One of the major challenges in the cancer therapy is the chemoresistance, which may play a critical role in cancer dissemination, relapse and patient clinical outcome. Retinoblastoma is the most common cancer affecting children eyes. Resistance to chemotherapy is only treated with enucleation in order to avoid brain metastasis. Among the diverse molecular mechanisms of resistance that cancer cells may activate, the upregulation of the oncogene survivin is recurrent. Survivin is the 5th member of the Inhibitor of Apoptosis Protein (IAP) family which its upregulation results in apoptosis inhibition, cell mitosis stimulation and DNA repair. Therefore, silencing survivin has been proposed as a promising therapeutic approach to overcome the anti-apoptotic phenotype resulting from the drug-induced survivin expression (1).
RNA interference (RNAi) is a naturally occurring mechanism responsible for gene regulation. The use of small interfering RNA (siRNA) is a powerful and convenient approach to selectively target and silence mRNAs through activation of the RNA-induced silencing complex (RISC). However, the efficacy of siRNA mainly relies on its delivery efficacy, limited by its highly hydrophilic nature and rapid nucleases degradation We have recently developed lipid nanoparticle able to protect siRNA in the systemic circulation and release it within the cell. These nanoparticles are based on a pH-sensitive molecular switch which triggers endosomal escape after cellular internalisation and promote cytoplasmic siRNA delivery. This mechanism demonstrated an efficient transfection efficiency in vitro and in vivo. (2). (Fig1)
In this project, we propose to deliver a survivin-targeted siRNA using pH-sensitive liposomes and evaluate the synergy with chemotherapeutics in the treatment of retinoblastoma.
Methods: The synthesis of the pH-sensitive lipid has been reported elsewhere (2). The pH-sensitive liposomes (SLP) (CSL:DSPC:Chol:DSPE-PEG2000 50:10:37.5:2.5 molar ratio) were formulated according to the lipid film hydration method and extruded through a 200 nm membrane. SLP were incubated with survivin-targeted siRNA (ThermoFisher, Ma, USA) for 30 minutes at 37°C 1200 rpm. Survivin silencing was performed in a panel of cancer cell lines, including retinoblastoma cells (Y79). Survivin downregulation was confirmed by qPCR and Western blot and its impact on cell cycle progression was evaluated by Flow Cytometry based on DNA content. Finally, we also measured the effect of different chemotherapeutics on survivin and caspase-3 expression in Y79 cells to rationally select a drug to perform viability studies with and without survivin-targeted SLP transfection.
Results: SLP was successfully formulated within nanometric size, homogeneous distribution and positive charge. Successful siRNA entrapment was also confirmed by SYBR Gold assay. Survivin-targeted SLP was able to downregulate survivin mRNA level to an extent comparable or higher than Lipofectamine RNAiMAX. Furthermore, survivin protein level was completely depleted in Y79 cells 96 hours followed transfection as confirmed by Western Blotting assay (Fig 2). Survivin downregulation also arrested Y79 cells at G2/M phase when compared to non-treated cells.
We also evaluated the mRNAs levels of survivin and capase-3 in Y79 cells following treatment with four chemotherapeutics currently used in the retinoblastoma protocol. We rationally selected carboplatin, which upregulated survivin and downregulated caspase-3 mRNA levels by 1.5-fold and 2-fold, respectively. In the viability study, survivin silencing significantly improved carboplatin effectiveness when compared with non-transfected cells.
Conclusion: Here we formulated pH-sensitive liposomes that were able to downregulate survivin in vitro, significantly improving carboplatin effectiveness in retinoblastoma cells. Those results may hold a promising clinical translational implication, encouraging further in vivo experimentation.
(1) ALTIERI, Dario C. Validating survivin as a cancer therapeutic target. Nature Reviews Cancer, v. 3, n. 1, p. 46, 2003.
(2) VIRICEL, W. et al. Cationic switchable lipids: pH-triggered molecular switch for siRNA delivery. Nanoscale, v. 9, n. 1, p. 31-36, 2017.