DEVELOPMENT OF PEG-COATED LIPOPLEXES WITH siRNA ANTI-E6/E7 ONCOPROTEINS TO BE INCORPORATED INTO MUCOADHESIVE HEC-SPONGES FOR THE TREATMENT OF HPV CANCERSTania Furst1, Anna Lechanteur2, Pascale Hubert2, Brigitte Evrard1, Géraldine Piel11Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liege, Liege, Belgium2 Laboratory of Experimental Pathology - GIGA Cancer, University of Liege, Liege, BelgiumE-mail : tania.furst@ulg.ac.be

1. INTRODUCTION• 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 the overexpression of oncogenes E6 and E7. In fact, the two encoded oncoproteins interact with tumor suppressor genes p53 and pRb and inactive them, which prevents apoptosis of tumor cells. These two oncogenes E6 and E7 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.

2. RESULTS AND DISCUSSION

Fig.2. (A) Gel retardation assay. The spot observed correspond to free siRNA and are compared to the control N/P=0 which is siRNA alone. The intensity decreases when N/P ratio increases meaning that siRNA is nearly totally encapsulated.

(B) RiboGreen® assay. Encapsulated siRNA is quantified at day 1, 2 and 6 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 and also until 6 days.(n=4)

3. CONCLUSION AND PERSPECTIVES

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2.1.a Preparation of cationic liposomes and siRNA-lipoplexes 2.1.c Preparation and characterization of PEG-coated lipoplexes

• The first purpose of this study is to develop a suitable vector, with specific siRNA anti-E6/E7, able to protect and transport it through the vaginal mucus and into the cytoplasm of cancerous cells. For this, cationic liposomes are used and by charge complementarities with negatively charged siRNA, lipoplexes are spontaneously formed. To be effective, these lipoplexes must have specific physico-chemical characteristics. Moreover, to facilitate the diffusion through the mucus, lipoplexes are peggylated by the addition of a lipid-PEG. • Secondly, lipoplexes will be incorporated into mucoadhesive hydroxyethylcellulose-gel, which will be freeze-dried to form a sponge, for topical treatment.

• 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 ± 5.6nm, with a low PDI=0.12 ± 0.03 and their zeta potential is +53.2 ± 6mV.

DOTAP/Chol/DOPE 1/0.5/0.5 Total lipid concentration 5mM

• Post-insertion technique: lipoplexes are peggylated by addition of DSPE-PEG2000 (in RNAse free water) at different percentages (from 5 to 50mol%of total lipids). The resulting mixture is vortexed for 15 seconds and maintained for 1 hour at 37°C.

2.1.b Characterization of lipoplexes• Z-average diameter and zeta potential of lipoplexes according to N/P ratios

• Encapsulation efficiency visualised with agarose gel (4%) electrophoresis and quantified using a Quant-iT™ RiboGreen® RNA assay • Evaluation of their physical stability

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Fig.3. Z-average diameter (nm), PDI and zeta potential (mV) of lipoplexes 1/0.5/0.5 at N/P=2.5 with different percentages of DSPE-PEG2000. (A) The diameter of the lipoplexes is ranged between 150 and 220nm, but from 25% of PEG the lipoplexes are too polydispersed (high PDI). (B) The zeta potential decreases when the % of PEG increases. (n=3)

2.2. Preparation of cellulose-derivative sponges• The sponges are obtained after freeze-drying of a homogeneous hydrogel composed by HEC and PEG400 in milliQ water.

Fig.4. RiboGreen® assay. siRNA’s encapsulation of lipoplexes at N/P=2.5 with 25% of DSPE-PEG2000, in comparison with lipoplexes at the same N/P ratio without PEG, at day 1,2 and 6 after their preparation. (n=4)

Lipoplexes 1/0.5/0.5 have good physico-chemical characteristics from the N/P ratio of 2.5. They present more than 95% of incorporation, a diameter at around 200nm and a positive zeta potential (+50mV). Moreover, up to 6 days after their preparation, there is no leakage of siRNA which means that lipoplexes have a high physical stability. Furthermore, after adding increased percentages of PEG, we observed a drop of zeta potential and 25% is the optimal to keep a slightly positive zeta potential (+15mV). We observed also that with 25% of PEG the encapsulation efficiency was as higher as without PEG. For the next perspectives, studies to verify the influence of pH variations, the diffusion through mucus and freeze-drying will be realized on these lipoplexes. Regarding the sponges, primary characterization has already been carried out, which allowed us to select the polymer (HEC) and the plasticizer (PEG400). The mucoadhesiveness, the rehydration speed, the hardness and the deformability will be quantified using an experimental design with a Texture Analyzer. Sponges containing lipoplexes will also be characterized.

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25%

Fig.5. (A) and (B) represent the HEC-sponges.

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Fig.6. are 2 examples of graph obtained with the Texture Analyzer to characterize the sponges. (A) is a cyclic compression test used to measure hardness and deformability (N). (B) is a mucoadhesion test. Adhesiveness of the sponge can be quantified.

(A) (B)

Fig.1. represents the Z-average diameter (nm) and zeta potential (mV) of lipoplexes formed at N/P ratios from 0 to 15 (100nM, 1000µl). From the N/P ratio of 2.5, the diameter is ranged between 180 and 220nm and the zeta potential remains constant at approximatively +50mV. (n=4).