Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes.
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2012
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We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown
effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells.
In furthering this line of research, the present study was focused on the interaction of siRNA to cationic
liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated
that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids
(DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the
negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome
membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change
in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA –
responsible for the characteristic bands of the A-form helix – and cationic liposomes were effectively
promoted. Factorial design coupled with response surface methodology was used to statistically analyze
the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of
siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation
(vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing
the gene knockdown effect of siRNA among the three variables, although all three are important. It was
concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized
the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting
in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the
preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of
siRNA.
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Vortex-mixing, Circular dichroism, Gene knockdown effect
Citação
BARICHELLO, J. M. et al. Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes. International Journal of Pharmaceutics, v. 430, p. 359-365, 2012. Disponível em: <http://www.sciencedirect.com/science/article/pii/S0378517312003523> Acesso em: 10 jan. 2017