Nanoheaters – Localized hyperthermia for precise gene delivery

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Publication . Ferreira, Daniela Filipa Cardoso; Baptista, Pedro; Fernandes, Maria Alexandra

Gene therapy relies on the precise transfection of nucleic acid effectors into cancer cells, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASO). Therefore, novel therapeutic approaches are widely needed to deliver silencing moieties with maximal trans- fection efficiency and minimal toxicity. This thesis explored the use of mild hyperthermia me- diated by gold nanoparticles (AuNPs) or magnetic nanoparticles (MNPs) to enhance nucleic acids delivery with spatiotemporal control over laser irradiation or magnetic modulation, re- spectively. First, the photothermal effect of AuNPs under visible light irradiation was verified, leading to enhanced cellular uptake. The potential of mild photothermy via AuNPs was demonstrated by effectively silencing the GFP gene in colorectal carcinoma cell line (HCT116) and breast adenocarcinoma cell line (MCF-7), with comparable gene silencing efficiency to commercial transfection reagent, but without cytotoxicity. Improving gene silencing strategies in 3D cell cultures is important since it provides in vitro models that closely resemble the in vivo tumor microenvironment (TME). Then, it was shown that mild photothermy mediated by AuNPs functionalized with ASO decreases c-MYC oncogene expression in HCT116 cells and 7-day spheroids, respectively. Localized magnetic hyperthermia mediated by immobilized MNPs on the cell mem- brane through bioorthogonal chemistry improves the transfection of anti-GFP in MCF-7 cells, with similar efficacy and less cytotoxicity compared to standard transfection reagent. Taking advantage of this approach, an effective IDO1 gene silencing was obtained through the trans- fection of siRNA in dendritic cells derived from an acute monocytic leukemia cell line (THP- 1). Moreover, the upregulation of pro-inflammatory cytokines IL6, TNFA, and IL12 genes and the downregulation of anti-inflammatory IL10 gene might contribute to a more immunogenic state in a TME context. In summary, this thesis highlights nanoparticle-mediated mild hyperthermia as a promising strategy for controlled and efficient nucleic acids delivery that might pave the way for improved gene therapy applications in more complex cancer models.

2025Tese de doutoramento

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Membrane-localized magnetic hyperthermia promotes intracellular delivery of cell-impermeant probes

Publication . Idiago-López, Javier; Ferreira, Daniela; Asín, Laura; Moros, María; Armenia, Ilaria; Grazú, Valeria; Fernandes, Alexandra R.; de la Fuente, Jesús M.; Baptista, Pedro V.; Fratila, Raluca M.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; RSC - Royal Society of Chemistry

In this work, we report the disruptive use of membrane-localized magnetic hyperthermia to promote the internalization of cell-impermeant probes. Under an alternating magnetic field, magnetic nanoparticles (MNPs) immobilized on the cell membrane via bioorthogonal click chemistry act as nanoheaters and lead to the thermal disruption of the plasma membrane, which can be used for internalization of different types of molecules, such as small fluorescent probes and nucleic acids. Noteworthily, no cell death, oxidative stress and alterations of the cell cycle are detected after the thermal stimulus, although cells are able to sense and respond to the thermal stimulus through the expression of different types of heat shock proteins (HSPs). Finally, we demonstrate the utility of this approach for the transfection of cells with a small interference RNA (siRNA), revealing a similar efficacy to a standard transfection method based on the use of cationic lipid-based reagents (such as Lipofectamine), but with lower cell toxicity. These results open the possibility of developing new procedures for “opening and closing” cellular membranes with minimal disturbance of cellular integrity. This on-demand modification of cell membrane permeability could allow the direct intracellular delivery of biologically relevant (bio)molecules, drugs and nanomaterials, thus overcoming traditional endocytosis pathways and avoiding endosomal entrapment.

2024-08-15Artigo científico

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Breaking the mold

Publication . Cordeiro, Sandra; Oliveira, Beatriz B.; Valente, Ruben; Ferreira, Daniela; Luz, André; Baptista, Pedro V.; Fernandes, Alexandra R.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Frontiers Media

Despite extensive efforts to unravel tumor behavior and develop anticancer therapies, most treatments fail when advanced to clinical trials. The main challenge in cancer research has been the absence of predictive cancer models, accurately mimicking the tumoral processes and response to treatments. The tumor microenvironment (TME) shows several human-specific physical and chemical properties, which cannot be fully recapitulated by the conventional 2D cell cultures or the in vivo animal models. These limitations have driven the development of novel in vitro cancer models, that get one step closer to the typical features of in vivo systems while showing better species relevance. This review introduces the main considerations required for developing and exploiting tumor spheroids and organoids as cancer models. We also detailed their applications in drug screening and personalized medicine. Further, we show the transition of these models into novel microfluidic platforms, for improved control over physiological parameters and high-throughput screening. 3D culture models have provided key insights into tumor biology, more closely resembling the in vivo TME and tumor characteristics, while enabling the development of more reliable and precise anticancer therapies.

2024Recensão

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Número da atribuição

2020.06599.BD