Among methods designed for siRNA delivery, one of the most promising is magnetofection, i.e., nucleic acid cell uptake under the influence of a magnetic field acting on magnetic nanoparticles that are associated with nucleic acid vectors. The major advantage of magnetofection is the possibility to localize nucleic acid delivery to an area where the magnetic field is applied. Moreover, the addition of a delivery vehicle, such as cationic polymer, can help to prolong the serum and intracellular half-lives of siRNA by improving pharmacokinetics and nuclease resistance, thus making the RNAi effect more therapeutically viable [1]. The INU-Cys-DETA copolymer here investigated, was constructed starting from inulin, a natural polysaccharide, by grafting diethylenetriamine (DETA) and cystamine (Cys). Grafting of DETA molecules lead to a copolymer bearing 1,2-diaminoethane moieties that, having a pKa between 8 and 10, confers the characteristic of polycation to the final structure. Amines can also interact with SPIONs surface [2], giving the possibility to form a hydrophilic coating that increase the in vivo circulation. Pendent Cys moieties contains a disulfide bond that is prone to be converted in thiol groups after proper reduction, while ensuring in the disulfide form the stability of the copolymer before use. Crosslinking of resultant pendent thiols, in the presence of siRNA and SPIONs, lead to the formation of tight and stable polymer-coated Super Paramagnetic Iron Oxide Nanoparticles named INU-Cys-DETA@SS@SPIONs/siRNA. Since several intracellular compartments such as cytosol, mitochondria, and cell nucleus contain a high concentration of glutathione tripeptide, which is 100 to 1000 times higher than that in the extracellular fluids and circulation, this strategy could elegantly resolve the contradictory requirements of efficient non-viral gene transfer agents, i.e. excellent binding and protection of nucleic acids in extracellular fluids and efficient release of nucleic acids inside the cells, by promptly release of siRNA in the cytosol upon redox-triggered disulfide cleavage to allow efficient mRNA knockdown. Obtained INU-Cys-DETA@SS@SPIONs and INU-Cys-DETA@SS@SPIONs/siRNA were characterized in terms of size and zeta potential and the existence of the polymeric coating and the chemical composition were confirmed by FTIR, quantitative iron determination, quantification of thiols and disulfide and determination of siRNA loading. In vitro studies on breast cancer cells, expressing luciferase gene, demonstrated that INU-Cys-DETA@SS@SPIONs had no cytotoxic effect in a wide range of concentration and that siRNA loaded systems are able to be taken up by cells and produce a satisfactory luciferase knockdown, increased by application of an external magnetic field. References. [1] Plank C., Zelphati O., Mykhaylyk O. Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Advanced Drug Delivery Review. 63, 1300-1331, 2011. [2] Licciardi M., Li Volsi A., Sardo C., Mauro N., Cavallaro G., Giammona G. Inulin-Ethylenediamine Coated SPIONs Magnetoplexes: A Promising Tool for Improving siRNA Delivery. Pharmaceutical Research. 32, 3674-3687, 2015.

Redox Responsive Inulin Copolymer Coated SPIONs for Tissue Targeted siRNA Delivery

Carla Sardo;Gennara Cavallaro
2016-01-01

Abstract

Among methods designed for siRNA delivery, one of the most promising is magnetofection, i.e., nucleic acid cell uptake under the influence of a magnetic field acting on magnetic nanoparticles that are associated with nucleic acid vectors. The major advantage of magnetofection is the possibility to localize nucleic acid delivery to an area where the magnetic field is applied. Moreover, the addition of a delivery vehicle, such as cationic polymer, can help to prolong the serum and intracellular half-lives of siRNA by improving pharmacokinetics and nuclease resistance, thus making the RNAi effect more therapeutically viable [1]. The INU-Cys-DETA copolymer here investigated, was constructed starting from inulin, a natural polysaccharide, by grafting diethylenetriamine (DETA) and cystamine (Cys). Grafting of DETA molecules lead to a copolymer bearing 1,2-diaminoethane moieties that, having a pKa between 8 and 10, confers the characteristic of polycation to the final structure. Amines can also interact with SPIONs surface [2], giving the possibility to form a hydrophilic coating that increase the in vivo circulation. Pendent Cys moieties contains a disulfide bond that is prone to be converted in thiol groups after proper reduction, while ensuring in the disulfide form the stability of the copolymer before use. Crosslinking of resultant pendent thiols, in the presence of siRNA and SPIONs, lead to the formation of tight and stable polymer-coated Super Paramagnetic Iron Oxide Nanoparticles named INU-Cys-DETA@SS@SPIONs/siRNA. Since several intracellular compartments such as cytosol, mitochondria, and cell nucleus contain a high concentration of glutathione tripeptide, which is 100 to 1000 times higher than that in the extracellular fluids and circulation, this strategy could elegantly resolve the contradictory requirements of efficient non-viral gene transfer agents, i.e. excellent binding and protection of nucleic acids in extracellular fluids and efficient release of nucleic acids inside the cells, by promptly release of siRNA in the cytosol upon redox-triggered disulfide cleavage to allow efficient mRNA knockdown. Obtained INU-Cys-DETA@SS@SPIONs and INU-Cys-DETA@SS@SPIONs/siRNA were characterized in terms of size and zeta potential and the existence of the polymeric coating and the chemical composition were confirmed by FTIR, quantitative iron determination, quantification of thiols and disulfide and determination of siRNA loading. In vitro studies on breast cancer cells, expressing luciferase gene, demonstrated that INU-Cys-DETA@SS@SPIONs had no cytotoxic effect in a wide range of concentration and that siRNA loaded systems are able to be taken up by cells and produce a satisfactory luciferase knockdown, increased by application of an external magnetic field. References. [1] Plank C., Zelphati O., Mykhaylyk O. Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Advanced Drug Delivery Review. 63, 1300-1331, 2011. [2] Licciardi M., Li Volsi A., Sardo C., Mauro N., Cavallaro G., Giammona G. Inulin-Ethylenediamine Coated SPIONs Magnetoplexes: A Promising Tool for Improving siRNA Delivery. Pharmaceutical Research. 32, 3674-3687, 2015.
2016
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11386/4728451
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