Aucun titre de diapositive

Aucun titre de diapositive

Tania Furst1, Anna Lechanteur1,2, Pascale Hubert2, Brigitte Evrard1, Graldine Piel1
Laboratory of Pharmaceutical Technology and Biopharmacy, CIRM, University of Lige, Belgium
Laboratory of Experimental Pathology, GIGA-Cancer, University of Lige, Belgium
E-mail: [email protected]

Human Papillomaviruses (HPV), most notably 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 the tumor suppressor genes p53 and pRb and inactive them, which prevents apoptosis of tumor cells.

The purpose of this project is to develop a suitable vector ( liposome ) able to protect and transport a specific siRNA through the vaginal mucus and
into the cytoplasm of tumor cells, in order to target and knockdown the mRNA encoding for E6-E7 oncoproteins. The physicochemical characteristics of
this vector must be optimal to be effective. Moreover, liposomes are peggylated because it has been demonstrated that coating nanoparticles with a
hydrophilic polymer like PEG, could improve the diffusion through the mucus.

3.2. Encapsulation of siRNA

3.1. Formulation and characterisation of liposomes
Lipids: - Cationic lipid DOTAP

- Fusogenic lipid DOPE
- Cholesterol

DOTAP/Chol/DOPE 1/0.5/0.5
Total lipid concetration = 5mM

To form lipoplexes, siRNA is added to liposomes at different molar
ratios (N/P) between the positive charges of the lipids (N) and the
negative charges of siRNA (P) in a nuclease free environment
[siRNA]=constant ; [liposomes]=increased
N/P : from 2,5 to 30
Liposomes 1/0.5/0.5

Fig.1: Hydration of lipidic film method

Free siRNA

The size of liposomes prepared in RNAse free water are between 150
and 200nm

3.3. Characterisation of lipoplexes : sizes and zeta potential
Analyse of the mean diameter (Z-average) and of the charge density of


Liposomes 1/1


Fig.2: Agarose gel electrophoresis (4%) is used to evaluate at which N/P ratio the
complexation occurs between the cationic liposomes and the negative siRNA.
For 1/0.5/0.5 and 1/1 liposomes, siRNA is nearly totally incorporated from the N/P ratio of 5

3.4. Peggylation of lipoplexes and characterisation
Lipoplexes are peggylated by the addition of DSPE-PEG2000 at 5, 10 and
20%mol of the total lipids by the post-insertion technique

Fig.3 and 4 represent the sizes and the zeta potential of 1/0.5/0.5 and 1/1 lipoplexes
according to the N/P ratio. At low N/P values, nanoparticles are large and heterogeneous.
From the N/P value 5, sizes become smaller and less polydisperse. Sizes are between 150
and 250nm and zeta potential is positive (+45mV)

Fig.5 and 6 represent the sizes and the zeta potential of peggylated lipoplexes at the N/P
ratio 5: DSPE-PEG2000 does not modify the size of lipoplexes while the zeta potential
decreases proportionally to the PEG percentage.

3.5. Transfection experiments with lipoplexes on SiHa cells
SiHa cells are HPV 16+ positive keratinocyte cells
Oligofectamine is a commercial transfection agent used as a positive control

Fig.7: Compared to Oligofectamine, the percentage of transfection with
unpeggylated lipoplexes is high (amore than 90%)
Fig.8: Peggylation of lipoplexes does not modify the percentage of
transfection. This percentage is around 90% for all the formulalions with the
3 different percentages of PEG


Resultant lipoplexes show good physicochemical characteristics from the N/P ratio of 5; a good encapsulation efficiency, an hydrodynamic diameter
between 150 and 250nm and a positive zeta potential. Moreover, the zeta potential decreases when DSPE-PEG 2000 is added and with a more significant
drop for 20% of PEG. The percentage of transfection on SiHa cells (HPV16+ cell line) is high for both unpeggylated and peggylated lipoplexes compared
to the Oligofectamine.
Other studies will be realized to confirm the stability over the time of the lipoplexes and to verify the influence of pH variations, protection from nucleases
degradation and contact with mucus.

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