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Every year, cancer causes millions of deaths around the globe, and researchers have been engaged to provide novel therapies for this threatening disease. However, this process is commonly hampered by failures in clinical trials. For this reason, efforts have been made to develop in vitro models capable of modelling the complex and heterogeneous tumour microenvironment, giving rise to organ-on-a-chip platforms. These devices have been paramount for further advancements in cancer research by providing a physiologically relevant platform with tumour organ-specificity. Organ-on-a-chip technology combines biomaterials, tissue engineering, oncology, and pharmacology in one device, making this a promising alternative to in vivo testing. This work reviews the progress made in organ-on-a-chip and multi-organ-on-a-chip models toward cancer research and provides an overview of the cells used, the 3D culture models developed and the materials selected to fabricate the microfluidic devices, Violeta Carvalho thank for her Ph.D. grant from FCT with reference UI/BD/151028/2021. Raquel O. Rodrigues thanks FCT for her contract funding provided through 2020.03975.CEECIND. This work has been also supported by the projects, EXPL/EMD-EMD/0650/2021, PTDC/ EMD-EMD/29394/2017 and PTDC/EEI-EEE/2846/2021 through the COMPETE2020, under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (FEDER) and by Fundação para a Ciência e Tecnologia (FCT). The authors also acknowledge the partial financial support within the R&D Units Project Scope: UIDB/00319/2020, UIDB/04077/2020, UIDB/04436/2020, UIDB/00532/2020.

Dissertação de mestrado em Biophysics and Bionanosystems, Nanossistemas de entrega de fármacos são utilizados para tratar uma vasta gama de condições, incluindo tumores sólidos. O objectivo da distribuição controlada de fármacos é encontrar o equilíbrio entre a terapia e os efeitos secundários reduzidos. No entanto, as células tumorais estão constantemente a evoluir mecanismos de resistência a fármacos, que sabotam e invalidam os tratamentos. Como consequência, está a ser investigado um nanocomposto híbrido orgânico/inorgânico para integrar sinergicamente capacidades de diagnóstico, terapêuticas e de modulação à resistência a fármacos numa só formulação. Neste estudo é desenvolvido um dispositivo de teranóstico, para administração de fármacos, controlado externamente. Os nanoveículos sólido lipidícos biocompatíveis foram carregados com nanopartículas magnéticas responsivas, doxorubicina, e verapamil, e foram caracterizados físico-química e funcionalmente. Os nanoveículos são esféricos e variam em tamanho de 150 a 200 nm, o que os torna ideais para administração, uma vez que este tamanho permite tirar partido da efeito de permeabilidade e retenção melhorada das células cancerígenas. Os nanoveículos apresentam um comportamento superparamagnético, tornando-os excelentes agentes de contraste T2 para o diagnóstico por ressonância magnética. Além disso, a encapsulação de nanopartículas de magnetite torna-o adequado para tratamentos de hipertermia, devido ao seu grande potencial de aquecimento se exposto a um campo magnético alternado. Uma avaliação in vitro dos efeitos das nanopartículas foi realizada em células de cancro colorrectal resistentes à doxorubicina (HCT-15) e em células de cancro da mama não resistentes à doxorubicina (HS-578T). Os ensaios de toxicidade confirmaram a biocompatibilidade do veículo e das nanopartículas magnéticas encapsuladas em concentrações clinicamente relevantes. Ao expor in vitro as nanopartículas carregadas com doxorubicina e verapamil, a um campo magnético, conseguiu-se um melhoria do efeito anti-tumoral demonstrando o potencial destas partículas para aumentar o efeito dos tratamentos clássicos através da combinação sinérgica de três fatores diferentes. Os sistemas de distribuição concebidos representam um passo em frente para o objectivo final de teranóstico personalizado. Os resultados obtidos são promissores para a utilização dos nanocompostos no diagnóstico precoce, e tratamento sinergético de tumores sólidos. Isto poderá resultar nos há muito desejados objetivos de menor toxicidade sistémica, eficiência dos agentes de contraste, maior eficácia anti-tumoral, e menos efeitos secundários na terapia do cancro., Promising nano drug delivery systems are now being used to treat a wide range of conditions, including solid malignant tumors. The goal of controlled drug distribution is to find the optimum balance between therapeutic gain and therapy-induced side effects. Nevertheless, tumor cells are constantly evolving multi-drug resistance mechanisms, which undermine and invalidate the treatments. Therefore, the use of a hybrid organic/inorganic nanocomposite to synergistically integrate diagnostic, therapeutic, and multi-drug resistance modulating capabilities into a clinically useful formulation is being investigated. An externally controlled theranostic multi-drug delivery device with increased antitumoral activity is developed in this study. Biocompatible solid lipid nanocarriers were loaded with response magnetic nanoparticles, doxorubicin, and verapamil, and were physico-chemically and functionally characterized. The nanoparticles are round and range in size from 150 to 200 nm, making them excellent for administration, since this size makes them ideal to take advantage of the enhanced permeability and retention effect of cancer cells. The nanocarriers showed a superparamagnetic behaviour, making them excellent T2 contrast agents in magnetic resonance imaging diagnosis. Furthermore, the inclusion of magnetite nanoparticles onto the delivery vehicle makes it also suitable for thermo-regulated treatments, due to its great heating potential when exposed to an alternating magnetic field. An in vitro evaluation of the magnetic solid lipid nanoparticles effects was performed in both doxorubicin resistant colorectal cancer cells (HCT-15) and doxorubicin non-resistant triple negative breast cancer cells (HS-578T). Toxicity assays confirmed the biocompatibility of the vehicle and the encapsulated magnetic nanoparticles at clinically relevant concentrations of iron ions. By exposing magnetic solid lipid nanocarriers loaded with doxorubicin, and verapamil to a magnetic field in vitro, a better anti-tumoral effect was achieved demonstrating the potential of these particles to enhance the effect of traditional treatments by synergistically combining the three effects (chemotherapy/multi-drug resistance modulating/hyperthermia). The designed delivery systems represent a step forward towards the final goal of personalised theranostics. The obtained results are promising for the use of the nanocomposites reported in early diagnosis, condition monitoring, and synergetic treatment of solid tumors. This could result in the long desired aims of lower systemic toxicity, contrast agent efficiency, increased anti-tumoral efficacy, and less side effects in cancer therapy.

Dissertação de mestrado em Ciências da Saúde, Cancer still ranks as a principal public health problem worldwide, being responsible for 9.9 million related-deaths in 2020. Breast cancer is the most commonly diagnosed cancer-type and the principal cause of cancer-related mortality in women, from which the triple-negative breast cancer (TNBC) subtype is the most lethal. TNBC treatment include conventional chemotherapy (e.g., doxorubicin/DOX), radiation and surgery, but response is poor, justifying the low survival rates of these patients. Thus, there is an urgent need for more specific and targeted approaches for early diagnosis, control, and treatment. The main objective of this thesis was the preclinical validation of a biocompatible theranostic probe activated in the tumor site through a specific external stimulus, a magnetic field. The nanosystem is based on magnetic solid lipid nanoparticles (mSLNs) loaded with a chemotherapeutic drug (DOX) and magnetic nanoparticles (Fe3O4), combining the synergistic effect of thermochemotherapy with non-invasive MR imaging. The combinatory effect of a dual treatment was explored in a first screening through IC50 determination in TNBC cells, where the temperature-responsive DOX-loaded mSLNs showed powerful anticancer properties against cancer cells, associated to a higher cytotoxic outcome in comparison to free DOX. Through more specific in vitro studies in 2D monolayer, mSLNs, as drug carriers, were able to inhibit cell proliferation and migration, in a time-dependent manner, being firstly internalized into the cell cytoplasm and, with an increase of incubation time, were able to release the drug into the cell nuclei. In the 3D spheroid model and the ex vivo Chick Chorioallantoic Membrane (CAM) model, the dual-treatment enhanced the penetration of the chemotherapeutic agent into the cancer cells, with improved therapeutic properties in comparison to free DOX. Then, in vivo studies were performed using an orthotopic xenograft NSG mice model. The unloaded nanoformulations demonstrated to be safe, without any detected toxic effects on animal-wellbeing and vital organ histology and promoted a T2-MRI contrast enhancement. Additionally, the DOX-loaded mSLNs demonstrated, within two weeks post-treatment, a significant capacity to inhibit tumor growth when combined with hyperthermia, in comparison to the other treatment conditions. In conclusion, the synergistic potential brought by the combination of active targeting with thermochemotherapy, provided by temperature-sensitive DOX-loaded mSLN, against TNBC cells, revealed theranostics capabilities, constituting a promising option as contrast agents for early diagnostics, coupled with an improvement in the therapeutic index in comparison to conventional chemotherapy., O cancro é ainda o principal problema de saúde pública mundial, tendo sido responsável por 9,9 milhões de mortes em 2020. O cancro da mama é o mais diagnosticado e a principal causa de mortalidade por cancro em mulheres, sendo o cancro da mama triplo-negativo (CMTN) o subtipo mais letal. O tratamento do CMTN inclui a quimioterapia convencional (por exemplo, doxorrubicina/DOX), radiação e cirurgia, mas a má resposta à terapêutica justifica as baixas taxas de sobrevivência dos pacientes. Assim, há uma necessidade iminente de abordagens mais específicas e direcionadas para o seu diagnóstico precoce, controlo e tratamento. O principal objetivo deste trabalho é a validação pré clínica de sondas teranósticas e biocompatíveis, ativadas no local do tumor através de um estímulo externo específico, um campo magnético. O nanossistema é baseado em nanopartículas magnéticas solido-lipídicas (mSLNs) carregadas com um fármaco quimioterapêutico (DOX) e nanopartículas magnéticas (Fe3O4), combinando o efeito sinérgico da termoquimioterapia com imagem não invasiva. O efeito combinado do tratamento duplo foi explorado numa primeira triagem através da determinação do IC50, onde as mSLNs responsivas à temperatura e carregadas com DOX exibiram propriedades anticancerígenas contra células do CMTN, associado a um resultado citotóxico superior ao da DOX livre. Através de estudos in vitro usando células em monocamada, as mSLNs carregadas com fármaco foram capazes de inibir a proliferação e migração celular, de forma dependente do tempo, sendo inicialmente internalizadas no citoplasma celular e, com o aumento do tempo de incubação, confirmou se a libertação do fármaco no núcleo. Com o modelo 3D de esferóide e no modelo ex vivo da Membrana Corioalantoide do embrião de galinha (CAM), observou-se que o tratamento duplo aumentou a penetração dos agentes quimioterapêuticos nas células cancerígenas, com um aumento significativo das propriedades terapêuticas em comparação com a administração da DOX livre. Estudos in vivo foram realizados usando o modelo ortotrópico em murganhos (NSG). As nanoformulações sem fármaco demonstraram ser seguras para o bem-estar do animal, sem serem detetados, por histologia, efeitos tóxicos em órgãos vitais, com um aumento do contraste em T2-RMI. Além disso, mSLNs carregadas com DOX demonstraram, até duas semanas após tratamento, uma capacidade de inibição significativa do crescimento tumoral através da combinação termoquimioterapêutica. Em conclusão, o potencial sinérgico da combinação termoquimioterapêutica das mSLNs sensíveis à temperatura e carregadas com DOX revelou ter capacidades teranósticas, constituindo uma opção promissora para o uso como agentes de contraste em diagnóstico precoce, associado a uma melhoria do índice terapêutico em relação à quimioterapia convencional., The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho, and International Iberian Laboratory of Nanotechnology. Financial support was provided by grants from the Foundation for Science and Technology (FCT) - project PTDC/BTM-SAL/31142/2017, ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by the project NORTE-01-0145- FEDER-000055, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF),and by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020.