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DEVELOPMENT OF PEG-COATED LIPOPLEXES WITH siRNA
ANTI-E6/E7 ONCOPROTEINS TO BE INCORPORATED INTO
MUCOADHESIVE HEC-SPONGES FOR THE TREATMENT OF HPV
CANCERS
Tania Furst1, Anna Lechanteur2, Pascale Hubert2,
Brigitte Evrard1, Géraldine Piel1 1Laboratory 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_at_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.

.

• 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.

2. RESULTS AND DISCUSSION
2.1.a Preparation of cationic liposomes and
siRNA-lipoplexes
2.1.c Preparation and characterization of
PEG-coated 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 5.6nm, with a low PDI0.12 0.03 and
  their zeta potential is 53.2 6mV.

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

2.1.b Characterization of lipoplexes

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

000
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. (n4).
Fig.3. Z-average diameter (nm), PDI and zeta
potential (mV) of lipoplexes 1/0.5/0.5 at N/P2.5
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.
(n3)
Fig.4. RiboGreen assay. siRNAs encapsulation of
lipoplexes at N/P2.5 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. (n4)

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

(A)

• Fig.2. (A) Gel retardation assay. The spot
  observed correspond to free siRNA and are
  compared to the control N/P0 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.
• (n4)

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.

(B)
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.
Fig.5. (A) and (B) represent the HEC-sponges.
3. CONCLUSION AND PERSPECTIVES
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.