•Human Papillomaviruses (HPV) such as HPV16 and HPV18 can induce cervical cancer. In this case, the two HPV E6 and E7 oncoproteins are essential players in order to immortalize keratinocytes by decreasing tumor suppressor genes (p53 and pRb). •Gene therapy is a promising strategy to treat cancer in order to decrease side effects against healthy cells. We focused on RNA interference (siRNA) to target mRNA coding for both HPV E6 and E7 oncoproteins (siRNA E6 and siRNA E7) and reactivate apoptosis. •To protect siRNA, to allow the diffusion into the cervical mucus and to cross the anionic cellular membrane, we use nanotherapy: siRNA is encapsulated in lipidic nanovectors to form LIPOPLEXES. In order to develop a topical treatment, polyethylene glycol are added on lipoplexes to increase the penetration through the cervico-vaginal mucus.

Flow cytometry assay

ASSOCIATION OF LIPOSOMES AND siRNA FOR A LOCAL TREATMENT OF CERVICAL CANCERAnna Lechanteur1,2, Tania Furst1, Brigitte Evrard1, Philippe Delvenne2, Géraldine Piel1, Pascale Hubert2

1Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liege, Liege, Belgium2Laboratory of Experimental Pathology, GIGA-Cancer, University of Liege, Liège, BelgiumE-mail : anna.lechanteur@student.ulg.ac.be

1. INTRODUCTION

2. RESULTS AND DISCUSSION

3. CONCLUSION / PERSPECTIVES

Association of liposomes and siRNA: physicochemical characteristics of lipoplexes

Figure 6 – siRNA anti-E6 and anti-E7 were both used to analyze reexpression of p53 by Western Blot. SiHa cells were transfected with Oligofectamine® and lipoplexes with siRNA at 100nM . Two days after transfection, they were harvested and analyzed. β-Actine antibody was used for normalisation.

Lipoplexes formulations cross safely cellular membrane and release siRNA into the cytoplasm

(All these results were obtained by Tania FURST, PhD student in LPTB)

p53 Western BlotFigure 4 – CaSki cells were treated with lipoplexes during 24 hours. After 2 hours of WST-1 reagent incubation, absorbance was measured with a spectrophotometer at 450nm and 691nm. Cytotoxicity was expressed as the mean percentage of cell viability relative to cells without any treatment. (n=4)

qRT-PCR assay

The association of liposomes and siRNA induced the formation of lipoplexes with appropriate physicochemical characteristics for a vaginal application. Lipoplexes containing siRNA 50nM and 100nM transfect easily HPV 16 positive cells line and induce a higher MFI than Oligofectamine® . The addition of PEG does not interfere with the transfection capacity. Lipoplexes with siRNA 50nM are safer than lipoplexes at 100nM. Cytotoxicity is proportional to siRNA concentration. Lipoplexes anti-E6 decrease the expression of mRNA E6. The addition of PEG reduce this extinction due to a steric hindrance. Lipoplexes anti-E6 and anti-E7 without PEG activate the re-expression of p53 protein on HPV 16 positive cells line. Percentage of PEG will be reduced and/or chain length will be modify to increase the efficacy of peggylated lipoplexes formulations.

Figure 5 – Lipoplexes anti-E6 were transfected in comparaison to Mock sample (siRNA scramble) and CaSki cells were analyzed two days after transfection. qRT-PCR assay (SYBR Green detection) have been performed to quantify mRNA E6 expression. The lowest extinction of mRNA E6 were observed with peggylated lipoplexes. (n=4)

Figure 3 – Transfection results were obtained by flow cytometry (FACS Canto II) with a siRNA labeled with Fluorescein one day after transfection. CaSki cells were treated with lipoplexes (at 50nM and 100nM) with or without peggylation and compared to Oligofectamine® . Percentage of transfected cells and MFI (Mean Fluorescence Intensity) were obtained. (n=4)

Cell proliferation WST-1 assay

DOTAP/Chol/DOPE 1/0,75/0,5

N/P %PEGZ-average diameter

(nm)

PDIzeta

potential (mV)

Liposomes 0 0 163.6 ±5.6 0.12 ± 0.0353.15 ±

6.0

Lipoplexes 2.5 0 219.9 ± 14.8 0.15 ± 0.05 48 ± 1.7

Lipoplexes-PEG 2.5 50 149.3 ± 8.6 0.28 ± 0.02 15.1 ± 0.5

•Three different lipids were choosen to form cationic liposomes: -Cationic lipid: DOTAP-Cholesterol-Fusogenic lipid: DOPE

Molar ratio: DOTAP/Chol/DOPE 1/0,75/0,5

Size Encapsulation assay

•Liposomes and siRNA were combined at a N/P ratio of 2,5 based on physicochemical characteristics.

•50% of polyethyleneglycol was added on lipoplexes (N/P 2,5) in order to increase the penetration through the cervical mucus.

Summary of the physicochemical characteristics formulations tested in vitro

Figure 1 A – Size and Zeta potential have been tested with Malvern Nano ZS. To modify the N/P ratio, concentration of siRNA is stable while the concentration of liposomes vary (n=4)

Figure 2 – Gel electrophoresis have been performed to determine the encapsulation of siRNA into the lipoplexes. N/P ratio of 0 correspond to the free siRNA.

Figure 1 B – Size and Zeta potential of lipoplexes as a function of the DSPE-PEG2000 percentage. (PDI = Poly Dispersity Index)(n=4)

NP

= 2,5

Surface chargeN/P ratio concept

Quantitative assay (Ribo Green® ) revealed that ±90% of siRNA was encapsulated in liposomes at a N/P ratio of 2,5.