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Heat up, silence on
Publication . Ferreira, Daniela; Asín, Laura; Idiago-López, Javier; Grazú, Valeria; Fuente, Jesús M. de la; Fratila, Raluca M.; Baptista, Pedro V.; Fernandes, Alexandra R.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Springer Science and Business Media Deutschland GmbH
Dendritic cells (DCs) are well-known antigen-presenting cells which have an important role in cancer immunomodulation due to the effective regulation of immune responses in the tumor microenvironment (TME). Indoleamine 2,3-dioxygenase 1 gene (IDO1) is upregulated in many types of cancers and associated with a poor prognosis, contributing to an immunosuppressive TME. IDO1 silencing in DCs is considered a promising new strategy in gene therapy owing to their capability to regulate T cells function and activation. This study focuses on the use of magnetic hyperthermia (MH) combined with bioorthogonal chemistry to promote siRNA transfection against IDO1 in THP-1-derived DCs. Magnetic nanoparticles (MNPs) functionalized with cyclooctyne moieties were attached by strain-promoted azide-alkyne cycloaddition to DCs membranes engineered to express artificial azide receptors. Upon the application of an alternating magnetic field, the MNPs generate heat and trigger the thermal disruption of the cell membrane. Results show that IDO1 gene expression decreases around 70% in THP-1-derived DCs, and that the MH-promoted transfection presents a silencing effect comparable to that attained with a gold standard Lipofectamine reagent, but with less cytotoxicity. Additionally, IDO1 silencing promotes the upregulation of mRNA levels of pro-inflammatory cytokines IL-6, TNF-α and IL-12A, and the downregulation of anti-inflammatory cytokine IL-10, providing a more immunogenic state which may lead to THP-1-derived DCs activation for future T cells antitumor response. Our findings reveal the potential of MH-mediated transfection to enhance the intracellular delivery of silencing moieties in cells difficult to transfect, such as DCs, as well as demonstrate the possibility of silencing IDO1 gene to overcome the immunosuppressive barrier imposed by the TME for cancer therapy.
Mild hyperthermia via gold nanoparticles and visible light irradiation for enhanced siRNA and ASO delivery in 2D and 3D tumour spheroids
Publication . Ferreira, Daniela; Fernandes, Alexandra R.; Baptista, Pedro V.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; BioMed Central (BMC)
Background: The delivery of therapeutic nucleic acids, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASO) into cells, is widely used in gene therapy. Gold nanoparticles (AuNPs) have proved to be effective in delivering silencing moieties with high efficacy. Moreover, AuNPs offer the possibility of spatial–temporal triggering of cell uptake through light irradiation due to their unique optical properties. Our study focuses on the use of AuNPs as improved vectorisation agents through mild photothermy triggered by visible light irradiation. This method promotes the transfection of oligonucleotides for gene silencing in 2D cells and more complex 3D spheroids. Results: Improving gene silencing strategies in 3D cell cultures is crucial since it provides more effective in vitro models to study cellular responses that closely resemble the in vivo tumour microenvironment. We demonstrate the potential of mild photothermy by effectively silencing the GFP gene in 2D cell cultures: HCT116 and MCF-7. Then we showed that mild photothermy could be effectively used for silencing the c-MYC oncogene transcript, which is greatly overexpressed in cancer cells. A decrease of 25% and 30% in c-MYC expression was observed in HCT116 2D cells and 7-day 3D spheroids, respectively. Conclusions: In summary, our findings offer a novel transfection approach for gene therapy applications in 2D and 3D tumour models. This approach is based on the use of mild photothermy mediated by AuNPs combined with visible laser irradiation that might pave the way for the spatial–temporal control of gene modulation.
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Fundação para a Ciência e a Tecnologia
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OE
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2020.06599.BD