1. DEVELOPMENT OF PEG-COATED LIPOPLEXES WITH siRNA ANTI E6/E7 ONCOPROTEINS FOR THE TREATMENT OF HPV CANCERS Tania Furst 1, Anna Lechanteur 1,2, Pascale Hubert 2, Brigitte Evrard 1, Géraldine Piel 1 1 Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liege, Liege, Belgium 2 Laboratory of Experimental Pathology – GIGA Cancer, University of Liege, Liege, Belgium E-mail : tania.furst@ulg.ac.be 1. INTRODUCTION 2. RESULTS AND DISCUSSION 3. CONCLUSION Human Papillomaviruses (HPV), particularly high-risk 16 and 18 genotypes, are responsible for chronic infection of keratinocytes of the uterine cervix mucosa. This infection is associated with the development of cervical cancer by overexpressed oncogenes E6 and E7. Those two encoded oncoproteins interact with tumor suppressor genes p53 and pRb and inactive them, which prevents apoptosis of tumor cells. They are attractive targets for the treatment of cancers induced by HPV. A topical treatment seems to be a promising strategy and has a great clinical interest. The purpose of this study is to develop an efficient delivery system, with siRNA anti-E6/E7, able to protect and transport it through the vaginal mucus and into cancerous cells. Cationic liposomes are used and by charge complementarities with negatively charged siRNA, lipoplexes are formed. To be effective, lipoplexes must meet specific physico-chemical characteristics. Moreover, to facilitate the diffusion through the mucus, lipoplexes are peggylated by the addition of a lipid-PEG. 2.1. Preparation of cationic liposomes and siRNA-lipoplexes Liposomes are prepared by hydration of lipidic film method Lipids : - Cationic DOTAP - Fusogenic DOPE - Cholesterol The mean particle size of empty liposomes is 163.6 nm (± 5.6), with a low PDI=0.12 (±0.03) and their zeta potential is +53.2 mV (±6). DOTAP/Chol/DOPE 1/0.75/0.5 Total lipid concentration 5,6mM 2.2. Characterization of lipoplexes Z-average diameter (nm) and zeta potential (mV) of lipoplexes according to N/P ratios (N and P = fixed) Fig.1. Lipoplexes are formed at N/P ratios from 0 to 15 (100nM, 1000µl). From the N/P ratio of 2.5, the diameter is ranged between 170 and 220nm (PDI < 0.1) and the zeta potential remains constant at +50mV. (n=4). Fig.2. (A) Gel retardation assay. The spot observed correspond to free siRNA (control N/P=0). Their intensity decreases when N/P ratio increases. siRNA is nearly totally encapsulated. (B) RiboGreen® assay. Encapsulated siRNA is quantified at day 0, 1 and 5 after the lipoplexes preparation. From the N/P ratio of 1.25, they present more than 95% of encapsulation. This percentage is constant until the N/P ratio of 15. The 3 curves overlap: encapsulation efficiency remains constant up to 5 days. (n=4) Encapsulation efficiency visualised with agarose gel (4%) electrophoresis and quantified using a Quant-iT™ RiboGreen® RNA assay Leakage of siRNA according to the time? 2.3. Preparation and characterization of PEG-coated lipoplexes Post-insertion technique: lipoplexes are peggylated by addition of DSPE-PEG 2000 (in RNAse free water) at different percentages (from 10 to 100mol%of DOTAP), vortexed for 15 seconds and maintained for 1 hour at 37°C. Fig.3. Z-average diameter (nm), PDI and zeta potential (mV) of lipoplexes 1/0.75/0.5 at N/P=2.5 with different % of DSPE-PEG 2000. (A) When the % of PEG increases, the diameter is ranged between 150 and 220nm. Up 50%, PDI 0.5. (B) Zeta potential decreases from +50mV to -20mV. (n=3) Fig.4. (A) RiboGreen® assay. siRNA’s encapsulation of lipoplexes 1/0.75/0.5 at N/P=2.5 with 50% of PEG in comparison with unpeggylated lipoplexes at the same N/P ratio, at day 0,1 and 5 after their preparation. Lipoplexes: mean % = 95.3% and lipoplexes-PEG: mean % = 89.6%. (n=4) (B) Z-average diameter of liposomes, lipoplexes and lipoplexes-PEG is constant for 5 days when they are stored at 4°C. (B) 50% (A) 2.5. Transfection efficiency (for more information see poster n° Anna Lechanteur) (A) (B) 2.4. Stability evaluation of lipoplexes (A)(B) Fig.5. Transfection experiment on CasKi cells (HPV16+). Lipoplexes and lipoplexes-PEG have a high % of transfection in comparison with oligoplexes (siRNA+Oligofectamine ® ). Mean Fluorescence Intensity (MFI) is higher for lipoplexes and lipoplexes-PEG than for oligoplexes. (n=3) Lipoplexes 1/0.75/0.5 have good physico-chemical characteristics and the optimal selected N/P ratio is 2.5 : > 95% of encapsulation and no leakage of siRNA up 5 days Z-average diameter of 219,9 nm (±14,8) and zeta potential of +48 mV (±1,7) When DSPE-PEG 2000 is added, the Z-average remains constant while zeta potential decreases  50% of PEG is optimal to obtain slightly positive lipoplexes with high density of PEG in their outer bilayer No significant difference in the encapsulation efficiency between lipoplexes and lipoplexes-PEG 50% and the encapsulation is constant for 5 days Empty liposomes, lipoplexes and lipoplexes-PEG 50% are stable: Z-average diameter is constant for 5 days The % of transfection is > 90% for both lipoplexes and lipoplexes-PEG 50%.  N/P ratio selected = 2.5