Polyplex based on polycarbonate polymers for an efficient delivery of HDAC5 and HDAC7 siRNA

RNAi therapeutics are promising therapeutic tools that have sparked the interest of many researchers. To meet the challenge of gene therapy and deliver small-interfering RNA (siRNA) into the cytoplasm of target cells, several barriers must be overcome. The most widely used polymeric vector is polyethylenimine (PEI) but its cytotoxicity limits its use in vivo. As a safe alternative, we designed and synthesized guanidium (PCG) and morpholino (PCM) functionalized biocompatible polycarbonate polymer with PTMC hydrophobic groups (figure 4) that is able to complex the siRNA with a significantly lower cytotoxicity than PEI. A RNA interference approach to anti-angiogenic therapy specifically targets the mRNA of histone deacetylases 7 (HDAC7). The shutdown of the HDAC7 protein disturbs the angiogenic process, making HDAC7 an attractive target that would directly interfere with the growth of cancerous tumors and metastasis development. Another possible target in the histone deacetylases family is HDAC5, controlling the cell-cycle progression and survival of human cancer cells. These results highlight the potential of polycarbonate vectors for future in vivo application as an anticancer therapy. Keyword(s): Polycarbonate, siRNA, Gene Delivery, Cancer. 1. EXPERIMENTAL METHODS 1.1 Incorporation of siRNA The ability of the polymer to complex siRNA was investigated by the Quant-iTTM RiboGreen® RNA reagent (Invitrogen, Life Technologies, Gent, Belgium). 1.2 Size and ζ-potential Polyplexes are prepared at a siRNA concentration of 100 nM. Z-average diameter and ζ-potential were determined using a Zetasizer Nano ZS (Malvern Instruments, UK). 1.3 Cellular Uptake The amount of siRNA taken up by cells was measured using flow cytometry. HeLa cells were treated for 3 hours with polyplexes formed with 100 nM in fluorescent labeled siRNA. 104 cells of each sample were analyzed using a FACSCalibur flow cytometer (BD Biosciences, FACSCalibur, USA). 1.4 HDAC5 and HDAC7 mRNA and Protein Expression HeLa cells were incubated 3 hours with HDAC5 or HDAC7 polyplexes. Then, cells were washed with DPBS and the medium was replaced by DMEM + 10% FBS and cells were incubated at 37°C for 48 hours. Quantitative real-time RT-PCR Quantitative real-time PCR was realized on a LightCycler® 480 (Roche, Mannheim, Germany) with βactine as control. Relative HDAC5 or HDAC7 mRNA expression compared to irrelevant conditions was analyzed using the 2 -ΔΔC T method. Western Blot analysis Equal amounts of proteins were resolved by SDS-PAGE. Membranes were probed with HDAC5 or HDAC7 antibody followed by horseradish peroxidase (HRP)-conjugated secondary antibodies, and developed by chemiluminescence detection. Detection of HSC70 protein was used as a loading control. 1.5 Cytotoxicity The cytotoxicity of polymers was determined using the WST-1 reagent (Roche, Basel, Switzerland). 2. RESULTS AND DISCUSSION 2.1 Incorporation of siRNA Increasing the ratio of PTMC-b-PCG-b-PCM polymer from 0 to 40 allows decreasing the free fraction of siRNA The Quant-iTTM RiboGreen® kit showed an incorporation of the siRNA reaching more than 90% from a N/P ratio of 20. 2.2 Size and ζ-potential Figure 1 shows the evolution of the z-average diameter and the zeta potential of PTMC-b-PCG-b-PCM polyplexes according to the N/P ratio. The optimal physico-chemical characteristics were reached from a N/P ratio of 40 where we obtained nanoparticles with a diameter of 157.1 nm and a zeta potential of 11.58 mV. Figure 1: z-average diameter and the zeta potential of PTMC-b-PCG-b-PCM polyplexes according to the N/P ratio. 2.3 Cellular Uptake With the polycarbonate polymer, we can observe in figure 2 an increase in the cellular uptake with the increasing N/P ratio, with 69% of transfected cells for N/P 20, 88% for N/P 30 and 95% for N/P 40, a similar result than PEI. Figure 2: Cellular uptake of polyplexes in HeLa cells was evaluated using flow cytometry (FACS). 2.4 HDAC5 and HDAC7 mRNA and Protein Expression The efficiency of polycarbonate polyplexes increase with the increasing N/P ratio to reach around 45% of relative mRNA expression at N/P 40, close to the PEI efficiency (figure 3A). This decrease of the mRNA expression was confirmed by Western Blot, showing the relative HDAC7 protein expression (figure 3B). Similar results were obtained with HDAC5. 2.5 Cytotoxicity No significant cytotoxicity was observed for the PTMC-b-PCG-b-PCM polymer. 3. CONCLUSION Synthetic guanidium and morpholino functionalized biocompatible polycarbonate polymers proved high ability to form polyplexes with low cytotoxicity compared to well-known PEI. PTMC-b-PCG-b-PCM polyplexes possess promising physicochemical characteristics and activity for a possible future use in gene therapy (figure 4). A B Figure 4: Chemical structure of PTMC-b-PCG-b-PCM polymer. References[1] Frère A., Kawalec M., Tempelaar S., Peixoto P., Hendrick E., Peulen O., et al. Impact of theStructure of Biocompatible Aliphatic Polycarbonates on siRNA Transfection Ability.Biomacromolecules, 16(3), 769-779, 2015.[2] Mottet D., Bellahcene A., Pirotte S., Waltregny D., Deroanne C., Lamour V., et al. Histonedeacetylase 7 silencing alters endothelial cell migration, a key step in angiogenesis.Circulation Research, 101(12), 1237-1246, 2007.[3] Peixoto P., Castronovo V., Matheus N., Polese C., Peulen O., Gonzalez A., et al. HDAC5 isrequired for maintenance of pericentric heterochromatin, and controls cell-cycle progressionand survival of human cancer cells. Cell Death & Differentiation, 19(7), 1239-1252, 2012.[4] Seow W.Y., Yang Y.Y. Functional polycarbonates and their self-assemblies as promising non-viral vectors. Journal of Controlled Release, 139(1), 40-47, 2009.