Application of Thermoresponsive PNIPAAM‑b‑PAMPTMA Diblock Copolymers in siRNA D...

• Author(s): Cardoso, Ana M. ; Calejo, M. Teresa ; Morais, Catarina M. ; Cardoso, Ana L. ; Cruz, Rita ; Zhu, Kaizheng ; de Lima, M. C. P. ; Jurado, A. S. ; Nyström, Bo
• Date: 2014
• Persistent ID: http://hdl.handle.net/10316/25534
• Origin: Estudo Geral - Universidade de Coimbra
• Subject(s): siRNA delivery; nonviral vector; cancer gene therapy; thermoresponsive copolymer; polyplex; cloud point
• Document Type: Article
• Language: English

Description

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).