Diélectrophorèse
La diélectrophorèse est définie comme le mouvement de matière causé par les effets de polarisation dans un champ électrique non uniforme (Pohl 1951; Pohl 1958). Ce phénomène ne nécessite pas l’utilisation de molécules globalement chargées, contrairement à l’électrophorèse. Tout dipôle possède une séparation de charges partielles plus et moins. L’application d’un champ électrique alternatif entraîne une orientation du dipôle selon ce champ. Une force sera alors créée et le dipôle sera déplacé. Dans le cas où la permittivité des particules en mouvement est supérieure à celle de milieu de suspension, les particules seront attirées dans les régions de fort champ électrique. Cependant, si la permittivité des particules en mouvement est inférieure à celle du milieu de suspension, les particules se dirigeront vers les zones de faible champ électrique.
Lorsque des vésicules géantes sont soumises à un champ électrique non uniforme, leurs dipôles induits s’alignent le long des lignes de champ. On observe donc une agrégation des vésicules les unes à la suite des autres sous forme de « collier de perles » le long des lignes de champ électrique.
Les conditions utilisées sont les suivantes (Bertsche and Zimmermann 1988) : - Signal sinusoïdal
- Fréquence f = 1,7 MHz
- Champ électrique E = 180 V/cm
Ces conditions permettent d’avoir des doublets de vésicules en environ 1 minute et des « colliers de perles » en environ 3-4 minutes, ce qui donne un temps suffisant pour faire la mise au point sur des doublets de vésicules.
Le générateur utilisé est un Agilent® 33220A, 20 MHz. Il est règlé sur 18 V pic à pic. Pour la chambre d’observation les électrodes sont espacées de 1 mm. Le champ électrique obtenu est alors de 180 V/cm. Plus le champ électrique est fort plus la diélectrophorèse se fait rapidement.
Fusion de GUVs
Après avoir mis deux vésicules en contact par diélectrophorèse, celle-ci est stoppée et une unique impulsion électrique de 100 µs de durée et d’environ 3 kV/cm est appliquée.
L’application de l’impulsion se fait de façon concomitante à l’arrêt de la diélectrophorèse grâce à un interrupteur permettant de passer d’un générateur à l’autre. Une série d’images espacées de 500 ms et de temps d’exposition 500 ms est enregistrée par l’intermédiaire de la caméra et envoyée sur l’ordinateur relié à cette caméra CCD. Les rayons des GUVs avant le processus de fusion et celui du GUV résultant sont mesurés en traçant les profils d’intensité de chacune des vésicules ; le diamètre est mesuré entre les deux pics d’intensité caractéristiques de la membrane (Figure 77).
Figure 77 : Principe du traitement d’image pour visualiser les rayons des GUVs avant et après fusion. L’image de gauche est l’image prise avant fusion, pendant la diélectrophorèse ; l’image de droite est prise après fusion des deux vésicules. Les profils d’intensité sont tracés le long des traits bleus présent sur les images. Les diamètres des GUVs sont mesurés entre les deux pics d’intensité caractéristiques de la membrane, représentés par les traits rouges.
Synthèse des vésicules de TriCat
(Soussan, Mille et al. 2008)Synthèse du tensioactif catanionique fluorescent FluoCat
L’acide 12-(N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)dodecaoique est additionné à un équivalent de N-hexadecylamino-1-deoxylactitol en solution dans l’eau pour conduire à une suspension hétérogène. Le milieu réactionnel est maintenu sous agitation à température ambiante jusqu’à ce que le réactif de départ, insoluble, disparaisse. Le produit de la réaction est récupéré après lyophilisation sous la forme d’une poudre orangée.
Synthèse des vésicules de TriCat
Une fois les tensioactifs synthétisés, les vésicules sont obtenues par quinze minutes de sonication d’une solution aqueuse contenant la poudre de tensioactif TriCat dans le cas des vésicules non fluorescentes, ou à partir d’un mélange de poudres 95% de TriCat pour 5% de tensioactif fluorescent en masse. Pour obtenir les vésicules, il suffit de se placer à une concentration supérieure à la concentration d’agrégation critique égale à 5.10-5 M dans l’eau. Leur taille est ensuite mesurée par DLS. Leur diamètre moyen est de 200 nm.
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Gene electrotransfer: from biophysical mechanisms to in vivo applications Part 1- Biophysical mechanisms
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