Descrição
Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and
dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to
avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at
evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3-
acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four
different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated
copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to
the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more
favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without
presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that
the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also
dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and
intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for
manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new
and promising siRNA delivery systems. This work was supported by the Portuguese Foundation for
Science and Technology and FEDER/COMPETE (research grants PTDC/QUI-BIQ/103001/2008, PTDC/DTP-FTO/
0265/2012, and Pest-C/SAU/LA0001/2013-2014) and by
the Norwegian Research Council, Project Number 190403.
A.M.C., C.M.M, and A.L.C. are recipients of fellowships from
the Portuguese Foundation for Science and Technology
(SFRH/BD/63288/2009, SFRH/BD/79077/2011, and
SFRH/BPD/46228/2008, respectively).
; Calejo, M. Teresa 
