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ASSOCIATION OF LIPOSOMES AND siRNA FOR A LOCAL
TREATMENT OF CERVICAL CANCER
Anna Lechanteur1,2, Tania Furst1, Brigitte
Evrard1, Philippe Delvenne2, Géraldine Piel1,
Pascale Hubert2 1Laboratory of Pharmaceutical
Technology and Biopharmacy - CIRM, University of
Liege, Liege, Belgium 2Laboratory of Experimental
Pathology, GIGA-Cancer, University of Liege,
Liège, Belgium E-mail anna.lechanteur_at_student.ul
g.ac.be

• 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.

Association of liposomes and siRNA
physicochemical characteristics of lipoplexes
(All these results were obtained by Tania FURST,
PhD student in LPTB)

• Three different lipids were choosen to form
  cationic liposomes

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

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

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.
Size
Encapsulation assay
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 (n4)
Surface charge
N/P ratio concept
Quantitative assay (Ribo Green ) revealed that
90 of siRNA was encapsulated in liposomes at a
N/P ratio of 2,5.
Summary of the physicochemical characteristics
formulations tested in vitro

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

DOTAP/Chol/DOPE 1/0,75/0,5 N/P PEG Z-average diameter (nm) PDI zeta potential (mV)
Liposomes 0 0 163.6 5.6 0.12 0.03 53.15 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
Figure 1 B Size and Zeta potential of
lipoplexes as a function of the DSPE-PEG2000
percentage. (PDI Poly Dispersity Index)(n4)
Lipoplexes formulations cross safely cellular
membrane and release siRNA into the cytoplasm
Flow cytometry assay
qRT-PCR assay
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. (n4)
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. (n4)
p53 Western Blot
Cell proliferation WST-1 assay
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. ß-Actine antibody was
used for normalisation.
Figure 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. (n4)

• 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.