Treatment of triple-negative breast cancer (TNBC) through externally triggered target less drug carriers

dezembro de 2021 UMinho | 2021 Universidade do Minho Escola de Medicina Mónica Alexandra da Costa Cerqueira Treatment of triple-negative breast cancer (TNBC) through externally triggered targetless drug carriers Treatment of triple-negative breast cancer (TNBC) through externally triggered target-less drug carriers Mónica Alexandra da Costa Cerqueira DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS This is an academic work that can be used by third parties as long as the internationally accepted rules and good practices regarding copyright and related rights are respected. Accordingly, this work may be used as provided in the license below. If the user needs permission to use the work under conditions not foreseen in the license indicated, he should contact the author, through RepositóriUM of University of Minho Atribuição-NãoComercial-SemDerivações CC BY-NC-SD https://creativecommons.org/licenses/by /4.0/ dezembro de 2021 Universidade do Minho Escola de Medicina Mónica Alexandra da Costa Cerqueira Treatment of triple-negative breast cancer (TNBC) through externally triggered targetless drug carriers Dissertação de Mestrado Mestrado em Ciências da Saúde Trabalho efetuado sob a orientação de Doutora Maria de Fátima Monginho Baltazar e de Doutor Manuel Bañobre-López iii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iv ACKNOWLEDGEMENTS Após a conclusão de mais uma etapa importante na minha vida, não posso deixar de agradecer às pessoas maravilhosas que tornaram tudo isto possível. Em primeiro lugar, agradeço à professora Fátima por toda a disponibilidade ao longo deste ano, dedicação, empenho e partilha de conhecimento. Ao Doutor Manuel Bañobre, gostaria de enaltecer toda a confiança que depositou em mim enquanto eu estava no INL, estando sempre disponível para me ajudar a crescer profissionalmente. À Doutora Marta Costa, não podia deixar de agradecer toda a dedicação, bondade, simpatia e empenho que teve comigo. Obrigada pelas conversas, sorrisos, apoio, ajuda e oportunidades que me proporcionaste. Ao Efres e à Raquel, ou “The Team”. Obrigada por todos os cafés e jantares fora de horas. À Céline, obrigada por toda a partilha de conhecimento e conversas intermináveis durante os longos dias no biotério. Às minhas Ana e Mafalda, obrigada, meninas, pelos almoços e companhia para o café, e pela linda amizade que criámos. As minhas meninas, Catarina, Rita, Natacha e Sónia, obrigada pelo lindo grupo de desabafo “We Social, We Share Meals”. Ju, Gui, Cunha e Cátia, vocês são os melhores amigos que poderia pedir. Por fim, quero agradecer aos meus pais e ao Vasco. Vocês são as pessoas mais importantes para mim, a minha família, o meu porto seguro. Obrigada por todo o apoio, pelas palavras na hora certa, pelo carinho e amor que transmitem. Por vocês e a vocês, SEMPRE. Amo-vos. Agradeço ao Professor Nuno Sousa, Presidente da Escola de Medicina da Universidade do Minho; Professor Jorge Correia-Pinto, Diretor do Instituto do Investigação de Vida e Saúde (ICVS); Professora Fátima Baltazar, Coordenadora do Domínio de Ciências Cirúrgicas, e à Professora Patrícia Maciel, Diretora do Mestrado em Ciências da Saúde pelo apoio institucional. FUNDING 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/BTMSAL/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-01-0145FEDER-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. v STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. vi ABSTRACT 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. Key words: Cancer, Triple-negative breast cancer, Theranostic, Nanoparticles, Hyperthermia vii RESUMO 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 préclínica 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, confirmouse 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. Palavras-chave: Cancro, Cancro da mama triplo-negativo, Teranóstica, Nanopartículas, Hipertermia viii INDEX Direitos De Autor E Condições De Utilização Do Trabalho Por Terceiros ............................................... iii Acknowledgements ............................................................................................................................. iv Funding .............................................................................................................................................. iv Statement Of Integrity .......................................................................................................................... v Abstract.............................................................................................................................................. vi Resumo............................................................................................................................................. vii Index ................................................................................................................................................ viii List Of Abbreviations And Acronyms .................................................................................................... xi List Of Figures .................................................................................................................................... xi LIST OF TABLES .................................................................................................................................... xiii 1| INTRODUCTION ........................................................................................................................... 1 1. Cancer ............................................................................................................................. 2 1.1 Breast Cancer ................................................................................................................. 4 1.2 Breast Cancer Subtypes .................................................................................................. 6 1.3. Breast Cancer Treatment ................................................................................................. 8 1.3.1. Doxorubicin Treatment .................................................................................................... 14 2. Nanomedicine In Cancer Theranostics ................................................................................... 15 2.1. Cancer Physiobiological Characteristics And Nanoparticle Features – Enhanced Permeability, Retention Effect And Tumor Nanoparticle Accumulation ........................................ 16 2.2. Magnetic Nanoparticles ................................................................................................. 18 2.2.1. Nanoparticle Composition .......................................................................................... 18 2.3. Solid Lipid Nanoparticles ............................................................................................... 19 2.3.1. Nanoparticle Composition .......................................................................................... 20 2.4. Magnetic Solid Lipid Nanoparticles ................................................................................. 21 2.4.1. Surface Modifications................................................................................................. 22 2.4.1.1. Pegylation For Nanoparticle Functionalization ......................................................... 23 2.4.1.2. Functionalization With Cell Penetrating Peptides ..................................................... 24 2.4.1.3. Diels-Alder Reaction ............................................................................................... 24 2.4.2. Biomedical Applications ............................................................................................. 25 2.4.2.1. Magnetic Resonance Imaging (Mri) As A Diagnostic Tool ......................................... 25 2.4.2.3. Drug Delivery ......................................................................................................... 28 xiii LIST OF TABLES Table I. Breast cancer statistics, in 2018. ................................................................................. 5 Table II. TNM anatomic parameters based on AJCC staging systems for breast cancer .............. 9 Table III. Classes of chemotherapeutic drugs. ....................................................................... 13 Table IV. Magnetic solid lipid nanoparticles (mSLNs) used in the present work. ........................ 33 Table V. Parameter of acquisition of relaxation time. .............................................................. 41 Table VI. IC50 values of the mSLNs and Doxorubicin for Hs578t and MDA-MB-231 cancer cell line .............................................................................................................................................. 49 Table VII. IC50 values of the mSLNs and Doxorubicin for Hs578t cancer cell line ....................... 53 Table VIII. Degrees of synergism for combination therapies...................................................... 55 Table IX. IC50 values of the mSLNs and Doxorubicin for THP-1 cancer cell line .......................... 58 1 1| INTRODUCTION 2 1. CANCER Cancer is a disease characterized by a set of heterogeneous disorders with different prognosis, origin sites, kinetics, and biomarkers (1). As a result of epigenetic, genetic modifications, and chromosomal aberrations combined with evolution and clonal selection, occurs the transformation of healthy cells into malignant phenotype with a deregulation on the pathways that coordinate the normal cell proliferation and homeostasis (Figure 1) (2–4). In cancer, there is an uncontrolled and rapid growth of aberrant and unfunctional cells with the capacity to metastasize to distant parts of the body (2). Lifestyle behaviors have a crucial influence on the onset of cancer, including physical inactivity, smoking, high solar exposure, poor diet, and reproductive changes ( e.g., later age of first child) (5,6). This heterogeneous disease still ranks as the principal public health problem worldwide (7), being the second leading cause of death worldwide, preceded by cardiovascular diseases (8). In 2020, it was registered 19.2 million of newly diagnosed cancer cases, and 9.9 million deaths from cancer-related disease in all countries (9). Moreover, according to the World Health Organization (WHO), in the next 2 decades, an increase of 60% of new global cancer incidence is expected (10). Regarding Portugal, the statistics revealed that, in 2018, it raised 58 199 of new cases and 28 960 of deaths (9). Figure 1. Transformation of normal cells into cancer cells. After the exposure to possible carcinogenic factors, genetic, epigenic modifications and chromosomal aberrations may occur. The cells that acquire evolutionary traits are selected obtaining the tumor. 3 Due to different types and subtypes that could be established within the same organ, cancer is considered a complex disease, where tumorigenesis is considered as a multistep event. In order to simplify the characteristics of cancer, in 2000, Hanahan and Weinberg defined an overall of set of traits acquired by cancer cells to survive, proliferate, and disseminate, as hallmarks of cancer. In this article, 6 hallmarks of cancer where proposed, including self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis (4). One decade later, the same authors revised and updated the concept of hallmarks of cancer, considering new ‘emerging’ characteristics: ability to evade immune destruction, to alter and reprogram cellular metabolism, inflammation through innate immune cells, and genomic mutation and instability (Figure 2) (2). More recently, the loss of differentiation was stated by Floor and coworkers (11). These hallmark capabilities are functional traits acquired by cancer cells through deregulation and reprogramming of different signaling pathways at distinct times during tumorigenesis, combined with the interconnections and crosstalk between the individual sub-circuits (2). By defects in signals responsible for growth, cell cycle, apoptosis, and cell migration, the cancer cells Figure 2. Hallmarks of cancer. Schematic illustration of the advantageous traits acquired by cancer cells essential for tumor growth and progression (adapted from [2]). 4 could decide their own fate, without maintenance of cell homeostasis. This dynamic system is a principal reason for the difficulty in reaching a specific treatment for cancer (12,13). 1.1 BREAST CANCER Breast cancer (BC) is the second most commonly occurring cancer worldwide (Figure 3), and it is considered the most diagnosed cancer-type and the principal cause of cancer-related mortality in women, with an estimation of 626 679 deaths in 2018 (14). In 2012, GLOBOCAN estimated an incidence of 1.7 million for both sexes, which already translates into high incidence. However, in 2018 the incidence number increased to 2.1 million cases (Figure 3), and for the year of 2040, 3 million new cases are estimated, pointing out the need to develop early-diagnosis strategies. Concerning the male population, male breast cancer has a rare occurrence, with less than 1% of all-cancer cases in men, and approximately 1% of all BCs around the world (15–17). Despite this, since 1975, male breast cancer mortality rate remained unchanged, with diagnosis in a late stage where it is observed large tumor size and lymph node involvement (17). The probability to occur a relapse is higher, as could be consulted on Table I where the prevalence of BC up to 5-years after the first diagnosis is expected (14). Contradictory to what would be expected, the highest BC incidence resides in countries with high Human Development Index (HDI), although the scenario in low HDI have propensity to aggravate until 2030, expecting an occurrence of 22.2 million of all-cancer cases (18). Figure 3. Worldwide statistics. Worldwide estimated number of cancer incidences, in both sexes, in 2018 [14]. 5 Table I. Breast cancer statistics, in 2018. Estimated incidence and prevalence for both sexes in Europe and United States of America (USA), in 2018 (14). *proportions by 100.000 In Portugal, for both sexes, breast cancer had an incidence and mortality of 7 041 and 1 864 cases, respectively, in 2020 (9). Due to its high incidence, breast cancer constitutes a concern to the scientific community, leading to increasing search for new targeted compounds with potential to diagnose and treat, known as theranostic agents. BC initiation and progression is associated to family medical history, environmental factors, hormonal therapy, genetical diseases, and inherited mutation in key genes, like BRCA1 and BRCA2 – two high-penetrance tumor suppressor genes, whose proteins have a role in DNA double-strand break repair (19,20). An efficient BC prevention enhances the probability of a better treatment response, emphasizing the need for an effective medical screening procedure, such as mammography (21). Screening by mammography is a gold standard method used for the identification of early BC in asymptomatic women, where small tumors that precede BC progression could be identified (22). A reduction of 19% in BC mortality was correlated to the implementation of mammography as a prevention tool, highlighting the correlation between an early diagnosis and a better prognosis (21). However, together with the possible harmful effects ( i.e ., pain, anxiety, and radiation risk), this method may produce false positives (benign tumors) and false negatives, promoting an inefficient detection of BC, being some cases detected in advanced stages of the disease (21–25). Early detection and monitoring of breast cancer are pointed out as main tool for the success of breast cancer treatment (26), in which diverse imaging techniques have emerged as major approaches for diagnosis and assessment of response to breast cancer therapy. Diverse studies indicated that magnetic resonance imaging (MRI), positron-emission tomography (PET), computed tomography (CT), ultrasound and single-photon emission computed tomography (SPECT) could be employed as efficient tools to detect BC, where mammography, ultrasound and MRI is the triple-modality currently applied (27–29). A study from 2015 indicated a higher propensity for MRI to detect pre-invasive and invasive cancers in women with a family Population Incidence Prevalence 1-year (prop.*) 3-year (prop.*) 5-year (prop.*) Europe 522 513 477 849 (124.3) 1 327 794 (345.5) 2 054 887 (534.7) USA 234 087 226 698 (137.4) 632 622 (383.4) 983 907 (596.3) 6 medical history and higher BC risk, comparatively to other imaging tools (28). Though, the imaging techniques have some associated drawbacks, such as being expensive (28), facing the need to use novel tools for cancer diagnosis. The use of the expression of biochemistry biomarkers ( e.g ., enzymes, hormones, mRNA expression profile, miRNAs, and exosomes) appears as new complementary diagnostic and therapeutic tools for patients with BC at various stages (27–31). 1.2 BREAST CANCER SUBTYPES BC is a complex disease where the epithelial cells lining the milk ducts are pointed as the origin spot. Breast cancer-type exposes a remarkable degree of interand intra-tumoral variability, revealing a high heterogeneity that is associated with either the anatomic site or phenotypically. Further understanding of BC heterogeneity, and the complex interplay between the tumor cells and different stroma elements will allow an appropriate diagnosis, a better insight of tumor progression, and the choice of an accurate targeted therapy (32). In the histological perspective (Figure 4), ductal carcinoma is the most common BC (50-70% of patients), followed by lobular carcinoma (5-15% of patients) (19,33). While histological stratification is a common practice in BC diagnosis, technological advances reveal further breast cancer complexity with different clinicopathological profiles (34). Through genetic sequencing and molecular analysis of breast cancer with the use of high-throughput gene expression microarray and next-generation sequencing, Perou et al. selected 496 genes using the criteria of significantly superior and minimum variations between different tumors and paired samples, respectively (35). The genes and samples were aggregated according to their similarity, where in the cluster analysis subset it is possible to observe two main branches, clinically described as ERpositive or ERnegative (estrogen receptor) (35,36). Moreover, using immunohistochemistry (IHC) and fluorescent in situ hybridization, the authors could also divide into more groups, PRpositive and PRnegative (PR, progesterone receptor) and can assess HER2 (human epithelial growth factor receptor 2) expression. Altogether, five different BC subtypes were identified: Luminal A, Luminal B, normallike, HER2-enriched, and Basal-like or Tripe-negative breast cancer (TNBC) (Figure 4) (19,33,35). However, this approach could not be enough to clearly identify the BC subtype, making the different aggressiveness of tumor subtypes indistinguishable. Taking this into account, St. Gallen 2013 (37– 39) classification also includes the assessment of proliferation-related genes, such as Ki67, 7 highlighting that subtypes which have high levels of Ki67 expression results in a worse prognosis (36). The medical goal is the correlation of different BC subtypes with clinical outcomes and response to targeted therapies. Differently from other BC subtypes, TNBC was distinguished into 6 subtypes by Lehmann et al. (37) through the analysis of the gene expressing of 386 tumors. The subtypes included two basal-like (BL1 and BL2), an immunomodulatory, a mesenchymal, a mesenchymal stem-like, and a luminal androgen receptor subtype. The accurate description of biological traits of BC subtypes enables i) a correct diagnosis of different BC subtypes, ii) a comprehension of the prognosis, risk of progression and occurrence of metastasis, and iii) a design of effective and targeted therapies, in terms of surgery, radiotherapy, or advanced targeted therapies, leading to higher disease-specific survival rates (38). Figure 4. Breast cancer anatomy and different subtypes. Overview of most common local to appear the BC and BC molecular subtypes (19,33,35). 8 1.3. BREAST CANCER TREATMENT The prescription of a BC treatment to individual patients is centered on diverse factors including general health state of the patient, stage, morphology, tumor grade, tumor size, presence of lymph nodes and metastasis, as well as expression of biomarkers (38). BC staging is a supportive tool for clinicians to determine the prognosis and design a specific treatment plan for individual patients. The American Joint Committee on Cancer (AJCC) staging systems for BC has assigned anatomical stages (0 to IV) based on tumor size (T), lymph node involvement (N) and the presence or absence of distant metastasis (M), forming the traditional TNM anatomic parameters (Table II) (40). 9 Table II. TNM anatomic parameters based on AJCC staging systems for breast cancer. TMN stratification based on tumor size (T), lymph node involvement (N) and the presence/absence of metastasis (M). Adapted from (40). Stage 0 ▪ Cancer cells stays within a duct; ▪ No evidence of surrounding fatty breast tissue invasion; ▪ Cancer have not spread to lymph nodes or distant sites. ▪ This is called ductal carcinoma in situ (DCIS), a non-invasive breast cancer. Stage IA ▪ Tumor is 2 cm across or less; ▪ Cancer has not spread to lymph nodes or distant sites. Stage IB ▪ Tumor is 2 cm across or less (or is not found); ▪ Micrometastases in 1 to 3 axillary lymph nodes (with a size not larger than 2 mm); ▪ Tumor has not spread to distant sites. Stage IIA ▪ Tumor is 2 cm or less (or is not found), has not spread to distant sites, and one of the following is applied: ▪ Tiny amounts of cancer are found in internal mammary lymph nodes; ▪ Cancer has spread to 1 to 3 lymph nodes under the arm and to internal mammary lymph nodes; ▪ Tumor is larger than 2 cm and less than 5 cm across, without spread to lymph nodes Stage IIB ▪ Tumor is larger than 2 cm and less than 5 cm across; ▪ It has spread to 1 to 3 axillary lymph nodes and/or tiny amounts of cancer are found in internal mammary lymph nodes. OR ▪ Tumor is larger than 5 cm across without grow into the chest wall or skin; ▪ Tumor has not spread to lymph nodes. Stage IIIA ▪ Tumor has not spread to distant sites, and: ▪ Tumor is not more than 5 cm across (or cannot be found), and it has spread to 4 to 9 axillary lymph nodes; OR ▪ Tumor is larger than 5 cm across, without any growth into the chest wall or skin. Moreover, it has spread to 1 to 9 axillary nodes, or to internal mammary nodes. Stage IIIB ▪ Tumor has grown into the chest wall or skin without metastasis into distant sites, and one of the following is applied: ▪ It has not spread to lymph nodes; ▪ It has spread to 1 to 3 axillary lymph nodes and/or tiny amounts of cancer are found in internal mammary lymph nodes; ▪ It has spread to 4 to 9 axillary lymph nodes or it has expanded the internal mammary lymph nodes. Stage IIIC ▪ Tumor at any size (or cannot be found), without metastasis at distant sites, and one of the following is applied: ▪ Tumor has spread to 10 or more axillary lymph nodes; ▪ Tumor has spread to the lymph nodes under or above the clavicle; ▪ Tumor involves axillary lymph nodes; ▪ Cancer has spread to both 4 or more axillary lymph nodes and into internal mammary lymph nodes. Stage IV ▪ Cancer can have any size and could spread to nearby lymph nodes; ▪ It has spread to distant organs or to lymph nodes far from the breast (most common organs are bone, liver, brain, or lung). 16 to their unique physical and optical properties, and chemical stability, nanoparticles can overcome various biological barriers that free drugs struggle with (92). Furthermore, surface functionalization can grant selectivity to drugs for specific body/organ/tissue targeting, and even for individual recognition and targeting of single cancer cells. Hence, the nanoparticle characteristics can benefit the bioactivity of the compounds through the reduction of the concentration needed for the same phenotypic outcome; potentially increasing their therapeutic index and pharmacokinetic properties, and altogether reducing their potential side effects on healthy tissues (84,92,93). Many nanoformulations have been investigated pre-clinically, yet only a minority have progressed to clinic stages (94). Currently, the approved by the United States Food and Drug (U.S. FDA) and European Medicines Agency (EMA) (20) nanoformulations include: Abraxane/paclitaxel (95), Doxil/doxorubicin (81), and ONPATTRO/Patisiran (96). These formulations try to fulfill the need to create new systems that improve drug selectivity and delivery, with greater efficiency and that help promote an early diagnosis of cancer. The development of cancer diagnosis tools is another research area in the nanomedicine field. WHO’s 2018 world cancer report predicted an increase of 60% in cancer incidence by 2040. Currently, early detection is the most effective way to increase the probability for successful treatment of most cancers. For TNBC, early detection is fundamental although screening is usually complicated due to low incidence of microcalcifications and to the presence of ductal carcinoma in situ which reduces the mammography effectiveness (97,98). These malignancies require a noninvasive, fast, and precise diagnostic system able to provide the location and the size of the tumors, in addition to the localization and size of other metastatic masses (99). Additionally, the monitorization of cancer-response to the treatment is a possibility, enabling an individual design of a later therapeutic strategy, implementing a more directed treatment with minimal side effects (100). 2.1. CANCER PHYSIOBIOLOGICAL CHARACTERISTICS AND NANOPARTICLE FEATURES – ENHANCED PERMEABILITY, RETENTION EFFECT AND TUMOR NANOPARTICLE ACCUMULATION Different nano-formulations, including lipid-based nanoparticles and polymer-based nanoparticles (Figure 8) (101–104), could reach the tumor through the leaky vascularization, a process called Enhanced Permeability and Retention (EPR) effect (105). In 1986, Matsumura et 17 al. found that proteins (15 – 70 kDa as molecular range) could preferentially accumulate in the tumor region for longer periods of time as a consequence of the enhanced vascular permeability with poor lymphatic drainage around the tumor (106). Over the last three decades, EPR effect became the principal dogma for the design of nanoparticles-based anticancer drug delivery in order to increase the efficiency of tumor therapy (105,107–111). Tumor growth requires a continuously supply of nutrients and oxygen to sustain the proliferation. The malignant cells secrete growth factors, as VEGF, and proteins, inducing the build of new blood vessels from the existing ones, a process called angiogenesis and well-known as one hallmark of cancer (Figure 2) (107–109). The rapid generation of new capillaries aggregated to a lack of basal membrane (vasculature supportive tissue) promotes the formation of abnormal vessels architecture with fenestration through the endothelium with 200 nM to 2 µM of diameter (107). The nanoparticles in circulation can easily extravasate to the tumor region through the fenestration located in the surrounded blood vessels, due to their characteristic small size (<200 nm) compared to the pore size (107,110). Conjugated to this enhanced permeability, an enhanced retention is also observed due to deficiency of the lymphatic system. Nanoparticles, characterized for a larger hydrodynamic size, are incapable to return to the surrounded capillaries, increasing the retention time in the tumor (106,107,109). Figure 8. Nanomedicine application on triple negative breast cancer. Different nano-formulations have been studied in order to improve the prognosis of hormone negative cancer and, consequently, offer a targeted treatment for TNBC (101–104). 18 EPR effect enables a higher drug accumulation in tumor compared to free drugs, decreasing the side effects associated to chemotherapeutic drug and increasing the efficiency of the tumor treatment (105,107–111). 2.2. MAGNETIC NANOPARTICLES Magnetic Nanoparticles (MNPs) are being widely studied now-a-days in many areas (such as in the biomedical field), because they offer a plethora of opportunities (97). Their physicochemical properties, superparamagnetic behavior, small size, and capability to promote biological interactions at the cellular and molecular level (97,112), allows MNPs to be employed as drug delivery systems (112,113), magnetic resonance imaging contrast enhancers (114), and hyperthermia inducers (115) for the treatment of cancer. The superparamagnetic properties of MNPs enable a degree of control through the application of an alternating magnetic field (AMF). Here, selective application of the AMF can force the MNP to generate local heat and promote direct tumor ablation and/or drug release into the desired region, ultimately avoiding invasive diagnostic and therapeutic techniques (116,117). MNPs have also the potential to be used as theranostic platforms in cancer therapy. A theranostic platform combines diagnostic and therapeutic capabilities in the same formulation, enabling an efficient tumor targeting, treatment, and monitorization of therapy response (or imageguided therapeutics; visualization of tissue images before, during and after the treatment) (117). This combination can help tailor the therapy requirements for each patient within an individualized therapeutic strategy design, with a greater probability of a positive outcome and, at the same time, reduced side effects (100). MNP performance is dependent on their composition, morphology, surface coating, and size of the inorganic core, all of which influence their in vivo behavior (97) and a potential toxicity (118). Studies performed in a mouse model with MNPs coated with DMSA (dimercapto-succnic acid) revealed accumulation in the liver, spleen, and lungs, without side effects (119). Hence, the functionalization of the formulations’ surface with targeted ligands can be a strategy to reduce toxicity in untargeted organs, whilst also increasing the therapeutic efficacy in targeted ones (118). 2.2.1. NANOPARTICLE COMPOSITION MNPs are composed with metallic, ferrites (MFe2O4, CoFe2O4, NiaZn(1-a)Fe2O4, MnaZn(1-a)Fe2O4), alloyed metallic (FeCo, alnico, and permalloy), and magnetic elements (hematite (αFe2O3), 19 magnetite (Fe3O4) and maghemite (γ-Fe2O3)) (118). The most commonly used nanoparticles in the biomedical field are superparamagnetic iron oxide nanoparticles (SPIONs), such as Fe3O4 and γFe2O3, which present high biocompatibility and lower toxicity compared to other metal structures ( e.g ., quantum dots, gold nanoparticles and carbon nanotubes (CNTs) may present lower biodegradation and body-elimination issues (97), together with increased cytotoxicity) (116,118). Surface functionalization is a critical requirement in order to increase their colloidal stability and biocompatibility (120). Both magnetite and maghemite present similar properties, whereas the structural differences between them are difficult to distinguish with the usual characterization techniques. Magnetite is characterized by a higher saturation magnetization compared to maghemite (92-100 versus 60-80 A.m2/Kg) (121). Due to their superparamagnetic behavior (particle sizes < 20-25 nm), both iron-based nanoparticles can be thermally activated through the application of an external magnetic field (122). Typically, these MNPs can be designed in two main configurations, (i) core-shell structures where the iron cores are coated with different organic or inorganic compounds ( e.g ., hydrophilic polymers, such as PEG or silica shells), (ii) the SPIONs are incorporated in different matrices ( e.g., polymeric, lipidic). The last configuration is in principle more suitable for theranostic purposes, since in addition to the drug encapsulation efficiency of the organic matrix the final system benefits from the physicochemical properties of the incorporated SPIONs (123). 2.3. SOLID LIPID NANOPARTICLES Over the last decades, the health-related scientific field has established and improved different drug delivery systems, including MNPs. As aforementioned, the nanocarrier’s success depends on their capacity to target a specific site, overcoming any anatomical barriers that block the action of the free drug, and ultimately allowing a selective, controlled, and sustained drug release. The composition of the nanocarriers (organic, inorganic, or hybrid), in addition to their sizes, shapes, and surface modifications can modulate their overall physicochemical properties (124). These modifications can further affect their biological properties, such as toxicity and stability, as well as the loading of drugs and nanoparticles in the system. One of these nanocarriers that have been extensively studied as a drug delivery system and which constitute the focus of this work are the solid lipid nanoparticles (SLNs) (125–128). 20 SLNs were first remarked in the early 1990s (129–132) as an upgrade of the alternative polymeric, inorganic and liposomic nanoparticles traditionally used until then as carriers (128). SLNs formulations are already approved by FDA or by GRAS (Generally Recognized As Safe), and are recognized as safe to be administered via different routes including intranasal, (133) inhalation (134), intravenous (135), subcutaneous (136), rectal (137), oral (138), ocular (139), and intramuscular (140). SLN design empowers the biodistribution and pharmacokinetics of the intended drugs, enhancing the drug treatment effectiveness by overcoming the MDR (141) which improves their bioavailability, protection from clearance by the reticuloendothelial system (RES), and controlling the drug release rate (125). Additionally, the possibility to modify the SLNs’ surface enhance the capability to overcome the biological barriers, to target the cancer cells with minimal side-effects (126), and decrease the initial rapid drug release, called “burst effect” (125) (major drawback of the controlled release system since they could expose the patient to drug overdose (142)). Identical to what happens with MNPs, coating the SLNs with PEG avoids the rapid immune system cell uptake of these nanocarriers and increase their circulation time (125,126,141,143). 2.3.1. NANOPARTICLE COMPOSITION SLNs nanocarriers are colloidal particles composed of a lipid matrix, solid at both room and body temperatures, and surfactants used as stabilizing and solvating agents. Different lipid and surfactant compositions can control the size, polydispersity, surface charge, stability, and drug release profile of the formulations (85). The selection of the lipid can also influence the biodegradability, stability, and embedment of the drugs and other elements (metals, dyes, etc.). Commonly, fatty acids, mono-, di-, and triglycerides, fatty alcohols, and waxes are used for the preparation of SLNs (144). The small size of the formulations (ranging from 10 to 1000 nm), the large surface-to-volume ratio, and the high drug encapsulation efficiency are the key advantages of the SLNs. SLNs enable the encapsulation of several drugs, both hydrophobic and hydrophilic (preferentially hydrophobic), that could be incorporated in four different ways: i) dispersed homogeneously in the lipid matrix, ii) dispersed throughout the shell, iii) incorporated in the core, and iv) dispersed on the external shell (86,145). As aforementioned, chemotherapeutic drugs are divided into different classes that could make difficult their encapsulation into nanocarriers. However, it is already demonstrated that SLNs efficiently incorporate different chemotherapeutic drug classes and could be used in the treatment of a wide range of cancers (82,146–149). 21 2.4. MAGNETIC SOLID LIPID NANOPARTICLES As aforementioned, SLNs present a broad variety of advantages for the treatment of cancer. Several research groups have focused on the development of these platforms trying to exploit and maximize these benefits (82,147–149). However somewhat surprisingly, the magnetic material incorporation in the SLNs was not explored until quite recently. Different metals and metal derivatives such as iron oxide, gold and gadolinium (84,89,93,100,150) have been incorporated in the nanoformulations, producing novel platforms with great potential in cancer therapy and tissue imaging. In particular, encapsulated iron oxide and gadolinium have been studied abundantly as magnetic delivery systems that can be guided to tumor regions and/or activated for controlled drug-release and cell ablation (magnetic hyperthermia) via an external magnetic field, or by pH changes (151–153). In particular, iron oxide nanoparticles are considered biocompatible and safe materials, that despite being able to cause some cytotoxic effects from the generation of ROS (reactive oxygen species) via Fenton reaction, which can lead to the damage of DNA, lipids, proteins, and carbohydrates (154). Magnetic solid lipid nanoparticles (mSLNs) represent a new class of functional nanoplatforms, which usually consist of inorganic magnetic nanoparticles and solid lipid nanoparticle mixtures, that have great applicability in the cancer field (155,156). The preliminary small size and high entrapment efficiency of the mSLNs managed to fuse the benefits of both types of nanocarriers (SLNs and MNPs) and overcome their independent application issues. mSLN shown an enhanced colloid and chemical stability and caused lower toxicity in vitro and in vivo models, compared to the MNPs alone, as described by Müller and colleagues (157). mSLNs synthesis can be achieved through different methodologies, including the emulsification dispersion-ultrasonic method (158), the emulsification-diffusion method followed by sonication (159), chemical co-precipitation (54,160), and the solvent evaporation technique (161). The characterization of the resulting mSLNs can then identify the structure of the formulation, where the magnetic nanoparticles can be embedded in the core and/or surface as described by several authors (159–162). On the one hand, a representation of magnetic nanoparticles embedded in the core can be seen in Figure 9, where the MNPs’ hydrophobic surface show chemical affinity by the lipid matrix to yield mSLNs. For the mSLN surface, different surfactants can be used during the synthesis to confer colloidal stability and solvation in water. 22 Altogether, mSLNs have demonstrated to be a promising tool due to their good biocompatibility (154–159), improvement of thermoresponsiveness compared to SLNs (156), efficiency in targeting tumors (156–161), and their high drug encapsulation efficiency. Furthermore, these nanosystems allow the application of magnetic hyperthermia as a mean to provide thermal therapy and control drug release (159–162), in addition to being used as MRI contrast agents (156). Still, there are only few studies involving tests in vivo , so highlighting the need to validate the performance of these nanocarriers in more biological complex systems. 2.4.1. SURFACE MODIFICATIONS The application of nanoparticles in the biomedical field is a challenging task where it is required to consider the nanoparticle biocompatibility and the capacity of cellular internalization by target cells (163,164). Normally, the systemically administered nanoparticles passively accumulate in the tumor tissue through the EPR effect (165,166). However, an effective nanoparticle biodistribution is intimately related with the nanoparticle-blood components’ interactions. Different biological factors could influence the efficient drug delivery from nanoparticles, including the mechanism of clearance from blood circulation. Thus, the immune system recognizes the Figure 9. Schematic structure of magnetic solid lipid nanoparticles (mSLNs) and their application in cancer theranostics. Due to the properties of magnetic nanoparticles (MNPs), mSLNs can be used for diagnostic purposes (e.g., MRI application), and cancer therapy, via magnetic hyperthermia. Moreover, magnetic hyperthermia in mSLNs offers an extra level of control over the drug release into the region of interest, ultimately increasing the cytotoxicity for cancer cells, in comparison with SLNs or MNPs alone. 23 nanoparticles (preferentially the hydrophobic ones) as foreign, activating macrophages from the mononuclear phagocyte system (MPS) (165–170). The biophysiological functionality is achieved through the functionalization of the nanocarrier, allowing its application as drug delivery system, contrast agent or theranostic probe (165–167). The surface functionalization is a strategy to prevent the nanoparticle phagocytosis through opsonization, increase the blood circulation time, improve the intracellular drug delivery and, consequently, reduce the drug dose administration (165,166). A broad range of ligands can be used to coat the nanoparticles’ surface, including surfactants, polymers, small molecules, peptides, antibodies, and oligonucleotides (163). 2.4.1.1. PEGylation for nanoparticle functionalization The hydrophobic surface of nanoparticles increase the opsonization which consecutively activate the MPS action reducing the bloodstream circulation (164,167,168). A physiochemical approach to avoid the final outcome is to coat the nanoparticles’ surface with hydrophilic polymers, such as polyethylene glycol (PEG), which resists interaction with blood components, imparting “stealth” properties to the nanoparticles (164,166–172). The biocompatible hydrophilic PEG possesses the capacity to minimize the binding of opsonins, limiting the immune system response and, subsequently, the coated nanoparticles remain longer periods of time in the bloodstream, enhancing the opportunity to passively accumulate in the desired region (166–173). PEGylation is widely used, as specific-targeting tool, in the cancer theranostic field. The surface modification of nanoparticles with PEG chains provides a targeted accumulation in tumor region due to EPR effect, with minimal side-effects for non-target cells (165,167). After extravasation from capillaries to the tumor environment, PEG chains are cleaved by the tumor-associated microenvironment components, such as matrix metalloproteinases (MMP), whose concentration is increased in tumor area (164,166). Thus, nanoparticle accumulation enables a clear recognition of tumor region for diagnostic proposes, as well as an improvement of targeted drug delivery, for treatment purposes (166–173). 24 2.4.1.2. Functionalization with Cell Penetrating Peptides In cancer treatment, the chemotherapeutic drugs place of action is in the intracellular region, as cytoplasm, nuclei, or mitochondria. However, due to impermeable barrier of the cell membrane, the therapeutic drugs can be barred from penetrating cells. As a strategy to overcome this problem, the conjugation between PEGylation and Cell Penetrating Peptides (CPP) has been used to enhance the drug intracellular uptake through tumor cells, taking advantage of a stealth effect (80,174). Surface functionalization with CPP is the mostly used platform to promote the lipid-based nanoparticle penetration into target cells, due to the positive charge of CPP (165,168,174–176). Peptides, as HIV-1 derived Transactivator of Transcription (TAT), are specific short peptides (less than 40 amino acids) capable to deliver cargos across the cell membrane, enhancing the drug delivery by endocytosis or direct penetration. Also, TAT-functionalization improves drug delivery, and the efficacy of a standard drug dose by overcoming drug resistance, associated with an attenuation of adverse effects (168,175,176). 2.4.1.3. Diels-Alder reaction The application of nanoparticles in the biomedical field is always accompanied with the purpose to enhance their biocompatibility, being crucial to employ chemical procedures that are reproducible and easy to manage (163,164). Click chemistry is an increasingly used technique with great applicability on surface functionalization of nanoparticles, specifically for a controlled-release systems (177,178). In 2001, Kolb and co-workers introduced the concept of “click chemistry” – a reaction different from the traditional ones – characterized by a generation of inoffensive final products and stable under biophysiological conditions (179). Due to the characteristics described by Kolb and colleagues, only some chemical transformations fulfill the criteria, in particular Diels-Alder (DA) cycloaddition characterized by their thermal reversibility is one of them (180). This one-step reaction forms a biocompatible cycloadduct without side products, considering an ideal click reaction (180–183). After reaching the pathological site, drug release occurs in response to microenvironment ( e.g ., pH) or external stimuli, as heat, radiofrequency, or ultrasound. DA reaction can be explored for nanoparticle surface decoration, enhancing the efficiency of drug release systems. Specifically, magnetite nanoparticles offer the possibility of activating DA thermal reversibility taking advantage of their magnetic properties to induce a temperature increase (181–183). Externally activated DAbased triggered mechanisms in conjugated systems enhance their applicability for delivery of 25 internalized molecules with a decline of toxic effects and promote the conjugation of theranostic tools with the controlled-release response – considered the next generation nanoparticles (182,183). 2.4.2. BIOMEDICAL APPLICATIONS 2.4.2.1. Magnetic Resonance Imaging (MRI) as a diagnostic tool WHO’s 2018 world cancer report predicted an increase of 60% in cancer incidence by 2040. Currently, early detection is the most effective way to increase the probability for successful treatment of most cancers. For TNBC, early detection is fundamental once screening being usually late and difficult (97). Detection of malignancy ideally requires a non-invasive, fast, and precise diagnostic system able to provide the position, size, and characteristics of the primary tumor, in addition to the localization and size of metastatic masses (99). MRI is a diagnostic tool used in the clinical setting for tumor detection and tracking, classified as a non-invasive, safe, and painless technique that uses magnetism and radio pulses to produce real-time images from different angles all around the body. The result is usually a clear depiction of soft tissues, including tumors (7,28,118). The technique is based on the nuclear magnetic resonance (NMR) principle, centering on the properties of some atoms to absorb energy in the form of radio waves when under a magnetic field, and consequently produce an electric signal (117,184). Such event causes a spin polarization that can induce a signal in a radio frequency coil that can then be detected by a nearby antennae/detector. Usually, hydrogen nuclei consisting of a single proton, are used to create the signals. Hydrogen is naturally abundant in all forms of life, and hence can be used to create a macroscopic polarization of hydrogen-rich tissues (rich in water and fat) (28,99). The pulses of radio waves excite the nuclear spin energy transition whilst the magnetic field gradients localize the polarization in space. After excitation, this technique measures the relaxation 1 time of the nuclei in the longitudinal ( T1 - spin-lattice relaxation), and transverse planes ( T2 - spin-spin relaxation) (99,117,185). The image formed here is dependent on the tissue’s local atomic density and association of hydrogen to other atoms (28). Therefore, the pulse sequence can generate different contrasts between tissues according to the different chemical environment of adjacent tissues. MRI enables rapid in vivo imaging acquisition with high spatial resolution (~100 µM), without exposing 1 The term relaxation refers to the movement of nuclear spins to their initial low-energy state when the radiofrequency wave is removed (118). 32 1. MAGNETIC SOLID LIPID NANOPARTICLES USED IN THE PRESENT WORK The nanoparticles studied for their anticancer potential in the present research work are based on the magnetic solid lipid nanostructures. Nanomedicine group at the International Iberian Nanotechnology Laboratory (INL) developed drug-loaded magnetic (mSLNs) from a commercially available vegetal wax, which was simultaneously loaded with a chemotherapeutic drug (DOX) and magnetic iron oxide nanoparticles (MNPs). Additionally, a surface functionalization was carried out using a mixture of two different responsive ligands. One of them was a temperature responsive PEGylated ligand that rendered mSLNs with stealthy properties, whereas the other was a cell penetrating peptide (CPP)-modified ligand. Thus, by local and external application of an AMF on the area of interest (magnetic hyperthermia, MH), a thermal-induced sheddable PEGylation is induced that exposes the CPP ligand and triggers on-site TNBC internalization through target-less magnetic carriers. Controls mSLNs formulations were also prepared to evaluate the different components in the final mSLNs design in order to accomplish the expected performance in vivo . The functionalized surface characteristics of the different samples used in this research are described in Table IV. 33 2. CELL CULTURE AND CELL LINES The human basal breast cancer cell lines Hs578t (basal-like), MDA-MB-231(basal-like), and the human macrophage cell line THP-1 were obtained from American Type Culture Collection (ATCC, Virginia, USA). Hs578t and MDA-MB-231 cell lines was cultured in Dubelcco’s Modified Eagle Medium, 4.5 g/L glucose (DMEM, Biochrom), while THP-1 cells were cultured in Roswell Nanoparticles Components Function DA-DOX-TAT-PEG-mSLNs (Nominated as EBR85) ▪ Cell-penetrating peptide (TATfunctionalization) ▪ PEGylation ▪ Diels-alder bond ▪ Carnauba Wax ▪ Tween89 (core-shell) ▪ Doxorubicin ▪ Magnetite (Fe3O4) Application on MRI diagnosis and TNBC therapy (theranostic properties), offering in a combination with a dual treatment capability (chemotherapy plus hyperthermia) DOX-TAT-PEG-mSLNs (Nominated as EBR86) ▪ Cell-penetrating peptides (TATfunctionalization) ▪ PEGylation ▪ Carnauba Wax ▪ Tween89 (core-shell) ▪ Doxorubicin ▪ Magnetite (Fe3O4) Control for Diels-Alder (DA) activation characterization DOX-TAT-mSLNs (Nominated as EBR52) ▪ Carnauba Wax ▪ Tween89 (core-shell) ▪ Doxorubicin ▪ Magnetite (Fe3O4) Control for ligands activity efficiency assessment DA-TAT-PEG-mSLNs (Nominated as EBR89 ▪ Cell-penetrating peptides (TATfunctionalization) ▪ PEGylation ▪ Diels-alder bond ▪ Carnauba Wax ▪ Tween89 (core-shell) ▪ Magnetite (Fe3O4) Control for doxorubicin (DOX) cell internalization and thermochemotherapeutic synergistic efficiency Table IV. Magnetic solid lipid nanoparticles (mSLNs) used in the present work. Schematic representation of the mSLN formulations used in this work, together with identification of their main function. 34 Park Memorial Institute (RPMI, Gibco), both supplemented with 10% heating activated Fetal Bovine Serum (FBS, Sigma-Aldrich) and 1% of antibiotic solution (Penicillin-Streptomycin, Gibco). Cells were grown in a humidified incubator at 37 ºC and 5% CO2. Sub-culturing was performed using 80% confluence culture flasks. Then, the cells were reaped by washing the T75 flasks with phosphate-buffer saline (PBS 1x) and detached from the flasks using trypsin (TryplexTM Express, Gibco) at 37 ºC. After the confirmation that the cells were no longer adhered, 10% FBS medium was added to the flasks to inactivate trypsin and then, cells were collected and centrifugated during 5 min, at 1200 rpm, 20 ºC. The culture medium was discarded and cells resuspended in medium to determine the number of viable cells, Trypan blue exclusion method (Trypan Blue Solution, 4%, Gibco) and Neubauer chamber were utilized. 3. 2D MONOLAYER CELL CULTURE 3.1. RESAZURIN ASSAY Resazurin dye has been used in several cytotoxicity studies as indicator of the cell viability. In this approach, aerobic respiration of metabolically active cells is responsible for the reduction of resazurin (blue compound) to resorufin (pink color). The resorufin fluorescence detected at 560 nm excitation / 590 nm emission is related to the number viable cells (199,200). A microplate reader (Biotek Synergy H1) was used to measure the fluorescence of the generated resorufin. 3.1.1. CELL VIABILITY ASSAY WITHOUT MAGNETIC HYPERTHERMIA APPLICATION To determinate the cytotoxicity effects of the studied compounds for both TNBC and macrophage cell lines, without MH application, the cells were plated in 96-well plates at a density of 5x103 cells/100 µL or 1x104 cells/100 µL, respectively, and incubated with supplemented medium. TNBC cells were allowed to adhere overnight at 37 ºC under 5% of CO2 environment and, in the next day, the medium was discarded and cells were treated with increasing concentrations of the medium containing the correspondent compound. Relatively to THP-1 cell line (grown in suspension), cells were treated with medium containing compounds on the same day that they were plated. The negative control was a group of cells not treated with the compounds. 35 Then, the cultured cells were incubated for 24, 46 and 72 h, and subsequently 10% of Resazurin (10x concentrated) was added in each well and incubated for 4 h. Spectrophotometrical measurements were performed in a microplate reader (Biotek Synergy H1), using 560 nm excitation / 590 nm emission sets. At least, three independent experiments were performed, each one in triplicate, and the statistical analysis software GraphPad Prism 7 was used to determine the IC50 (half-maximal inhibitory concentration) values of each compound, applying a sigmoidal doseresponse (variable slope) non-linear regression after logarithmic transformation. The therapeutic gain (TG) was calculated in comparison with the IC50 value of free DOX when no hyperthermia was applied (Equation 1). 𝑇𝐺 (%)=100 𝑥(𝐼𝐶50𝐷𝑂𝑋 𝐼𝐶50𝑚𝑆𝐿𝑁𝑃 -1 ) Equation 1 3.1.2. CELL VIABILITY ASSAY WITH MAGNETIC HYPERTHERMIA APPLICATION The overheating characteristic of MH treatment is widely used as an adjuvant in cancer therapy (193,194). With the application of an AMF, MNPs can locally transform the electromagnetic energy into heat, increasing the local cancer cell’s temperature and resulting in tumor growth inhibition (193). To select the frequency and amplitude of the external AMF, it is necessary to consider the Brezovich limit ( H0f < 4.85x108 A.m-1.s-1), which specify the upper limit for the magnetic field that can be applied to human tissues avoiding undesirable heating of surrounded tissues (201). Firstly, the sample is placed inside a helical induction coil, where the selected amplitude of the AMF is achieved. Therefore, an alternated current flow along the coil changing, at chosen frequency, the magnetic flux. The equipment is also composed by a temperature sensor (optical fiber), connected to a computer that records the temperature changes along time (202,203). In this work, the cytotoxicity effect of the synergistic thermal treatment was evaluated by performing the cell viability assay after MH application, where Hs578t cells were seeded in a dedicated Petri dish (divided in 4 compartments) at a density of 2.5x104 cells/500 µL. Cells were incubated overnight at 37 ºC under 5% of CO2 atmosphere. Thereafter, the medium was removed and the cells were treated with increasing concentrations of the medium containing the mSLNs of interest and subjected to an AMF at a field and frequency of 23 mT and 174.5 kHz, respectively, 36 for 1 h, in a NAN201007 Live Cell Exposure module from NanoTherics. For control purpose, one group of cells did not receive further treatment. Cells were incubated again for 24, 48 and 72 h and, after each time-point, 10% Resazurin was added to each compartment and incubated for 4 h. Then, the solution from each compartment was transferred onto a 96-well plate and spectrophotometric analysis was performed in a microplate reader, using 560 nm excitation / 590 nm emission sets. At least, three independent experiments were performed, each one in triplicate, and the statistical analysis software GraphPad Prism 7 was used to determine the IC50 values of each compound, applying a sigmoidal doseresponse (variable slope) non-linear regression after logarithmic transformation. The activity gain (AG) by hyperthermia was calculated by firstly normalizing to the DOX IC50 value (without hyperthermia application), and then comparing the DOX IC50 with the mSLNs IC50 after hyperthermia application (Equation 2). 𝐴𝐺 (%)=100 𝑥 (𝐼𝐶50 𝐷𝑂𝑋 𝐼𝐶50 𝑚𝑆𝐿𝑁𝑃 𝐼𝐶50 𝐷𝑂𝑋 & 𝑀𝐻 𝐼𝐶 50 𝑚𝑆𝐿𝑁𝑃 & 𝑀𝐻 ⁄ −1) Equation 2 The degree of synergism of the dual-treatment was calculated using the Equation 3, where MHT is the MH and Chemo is the chemotherapy. For a α equal to 1 the treatment applied had an additive effect; α<1, the dual-treatment is antagonist, and α>1 represent a synergistic effect for the dual-treatment. 𝛼= 𝐼𝐶50 𝑀𝐻𝑇.𝐼𝐶50 𝐶ℎ𝑒𝑚𝑜 𝐼𝐶50 𝑀𝐻𝑇+𝐶ℎ𝑒𝑚𝑜 ⁄ Equation 3 3.2. CONFOCAL MICROSCOPY IMAGING To follow the cellular phenotype of breast cancer cells upon their interaction with mSLN formulations, confocal microscopy was used. Confocal microscopy offers several advantages face to conventional optical microscopy where, the principal point is the elimination, or reduction, of background “noise” from the focal plane (204). The conventional optical microscopy uses a beam of light to directly illuminate the entire field of the sample, being all parts of the sample excited at same time. However, the final image is composed by both in-focus light at the focal plane of the lens and out-of-focus light coming from 37 other focal planes, promoting a decrease of signal to noise (204,205). In contrast, the confocal microscopy uses a pinhole at the image plane to prevent that the out-of-focus light does not reach the detector, scattering the other light. Though, information about a unique point of the sample is provided and therefore, it is necessary to move the specimen or alter the exciting light to obtain the final image [195]. In this way, two-dimensional images could be acquired plane to plane, at different depth, enabling the three-dimensional reconstruction by using a microscopy deconvolution software (z-stack). This technique allows the detection of different sections of the sample due to dyes that turn fluorescent when stimulated with light, named fluorophores. By using a specific wavelength to excite the specimen, it enables the excitation of specific fluorophores (206). In this study, the confocal microscope LSM780 from Zeiss was used to visualize the interactions between Hs578t live cells and the treated compounds. On the day before, 6x103 cells were seeded in Petri dish divided in 4 compartments, and then incubated overnight at 37 ºC under 5% CO2. Thereafter, the medium was removed and the cells were treated with medium containing the compounds. The cells were again incubated for 2 different time points, 2 h and 6 h. After the incubation time, the cells were washed with 1x PBS and Hoechst 33342 was added (1:1000, cat. no. ab139481, Abcam). The cells were incubated for 10 min, washed again with 1x PBS, and then DMEM phenol red-free was added. The inherent fluorophore (DiO) incorporated in mSLNs membranes permitted to track the designed nanoparticles, and the DOX inherent fluorescence was followed and co-localized with a nuclear stain (Hoechst 33342), using the excitation laser of 488 nm for DOX and DiO, and 405 nm for Hoechst 33342. 3.3. CELL PROLIFERATION ASSAY The cell proliferation is a colorimetric immunoassay evaluated using 5-bromo-2’-deoxyuridine (BrdU) (Cell Proliferation ELISA, BrdU, Roche®). BrdU is a pyrimidine analog which could be incorporated into cells DNA during the synthesis process. According to manufacturer's instructions, Hs578t cells were plated in 96-well plates, in triplicate (8000 cells/well), and allowed to adhere overnight in complete DMEM medium, at 37 ºC in a 5% CO2 humidified atmosphere. After medium removal, the adherent cells were treated with respective EBR85, EBR89 and DOX at IC50 and 1⁄2 IC50 dosages for 24 and 48 h of incubation. The controls were treated with a complete DMEM medium. After incubation, BrdU diluted in 1% FBS culture medium was added to the cells (final BrdU concentration of 10 μM) and reincubated 38 for 6 h. During this incubation step, BrdU was incorporated (in thymidine place) into DNA of proliferative cells. After BrdU labeling, cells were fixed, and DNA was denatured through incubation with 200 μL of FixDenat solution for 30 minutes at room temperature. After removal of this solution, 100 μL of Anti-BrdU-POD antibody (1:100 dilution) were added, which will bind to recently incorporated BrdU in the cells’ DNA. Following 90 min of incubation at room temperature, the wells were rinsed three times with PBS 1x. Next, 100 μL of substrate solution (tetramethyl-benzidine) was added at room temperature for 5 min until color development. The colorimetric reaction was stabilized by adding 25 μL/well of 1 M H2SO4. The reaction product was quantified by measuring the absorbance at 450 nm (Thermo Scientific Varioskan® Flash). A blank control was used in each experimental time point, without cells. Percentage of cell proliferation (normalized to control condition) from at least three independent experiments was evaluated with the GraphPad Prism 7 software. 3.4. WOUND-HEALING ASSAY Cell migration ability was appraised by the wound-healing assay in order to mimic the migration event that occurs in vivo. The basic steps consist in performing a scratch in a confluent well plate, simulating a wound, capturing images at the beginning of the experiment and at different regular time-points to, at the end, compare the images and quantify the cell migration rate in different treatments (207). Hs578t cell line was seeded in 6-well plates at a density of 9.0x105 cells/well/2 mL and incubated overnight at 37 ºC under 5% of CO2 atmosphere. At time point zero, culture medium was removed and two scratches were executed with a 200 µL pipette tip on the cell monolayer. The cell debris were detached by gently washing with 500 µL 1x PBS, and the cells treated with respective compounds (free DOX, EBR85, and EBR89) at the respective IC50 value or medium only for control purposes, for 48 h. At time-points of 0, 12, 24, 36 and 48 h of treatment, eight specific “wounded” areas per condition were photographed at 100x magnification by phase contrast microscopy (Olympus® IX51). The migration distances from at least three independent assays were assessed using the beWound - Cell Migration Tool (Version 1.5), and the percentage of cell migration rate normalized to the control condition was evaluated with the GraphPad Prism 7 software. 39 4. 3D BIOMODELS (SPHEROIDS) 4.1. DEVELOPMENT OF SPHEROIDS Although the 2D monolayer cell culture offers many benefits, due to their simple procedure, they could not mimic the real cell microenvironment, resulting in a poor reproduction of the nanoparticle-cell interactions. The cells normally live in 3D environment essential for their metabolism, growth, and cell-cell interactions. For drug screening, the use of 3D cell culture, as spheroids, is important since it considers the cell morphology, proliferation, waste production, and drug uptake. Moreover, cellular response to drug therapies in 3D culture displayed results more similar to what happens in vivo (208,209). In the current work, Hs578t spheroids were obtained from Hs578t cell line culture. Firstly, 1x104 cells/100 µL were seeded in 96-well plates, containing 100 µL of agarose 1% in each well. Then, the plates were centrifuged at 1600 rpm for 30 min to allow the cells to settle by gravity. Subsequently, the plates were incubated at 37 ºC under 5% CO2. For 1 week, the spheroids’ medium was replaced every 2 days. On the second week, spheroids were seeded in a Petri dish with 4 compartments containing agarose 1% (4 or 5 spheroids per quadrant), forming the polyspheroids, and the medium was changed every 2 days. 4.2. EVALUATION OF 3D POLYSPHEROID GROWTH In order to evaluate the response of Hs578t polyspheroids to a single (only DOX-containing mSLNs) and dual therapy (DOX plus MH), spheroid growth was assessed along 3 weeks and compared to free DOX treatment. After spheroid formation, treatment was conducted by adding 10 µM of the corresponding compound. Then, the Petri dishes planned for dual treatment were subjected to AMF at 23 mT of field and 174.5 kHz of frequency, for 1 h. The same treatment was performed every 2 days for 1 week. Thereafter, during the following 2 weeks, the medium was changed every 2 days. To assess polyspheroid growth, photographs were taken every 2 days, representative of each study condition, starting 3 days before the beginning of the treatment. Through the photographs, and using the RStudio program, it was possible to measure the spheroid daily growth, by firstly normalizing the growth per spheroid (Equation 4), 𝐺𝑛 (%)=100 𝑥 𝐴𝑟𝑒𝑎𝑑𝑎𝑦 𝐴𝑟𝑒𝑎𝑑𝑎𝑦=1 Equation 4 40 where the Areaday is the spheroid area corresponding to each day and the Areada=1 is the measured spheroid area at the beginning of the treatment. Then, using the Equation 5, we obtain the normalized daily growth enabling the comparison of different conditions tested independently of time. 𝐷𝐺𝑛 (%)=∑𝐺𝑛 𝐷𝑎𝑦𝑠= ∑𝐺𝑛 19 Equation 5 4.3. MAGNETIC RESSONANCE IMAGING (MRI) STUDIES The magnetic properties of iron oxide nanoparticles have made them interesting probes in MRI. These properties enable the easy co-localization of the MNP when passively or selectively accumulated in tumors, whilst in turn they can simultaneously provide a therapeutic effect ( i.e . drug delivery, MH) – theranostics (119). In order to evaluate the T2 -MRI contrast enhancement capability of the developed mSLNs, samples were placed inside the scanner bore, where an oscillate magnetic field is created, promoting the alignment of the protons in same direction B0 . By a set of gradient coils, the radiofrequency magnetic field is switched from on to off, at an appropriate resonance frequency, and the proton alignment alters, realigning with the magnetic field. As a result of the signal coming from the excited atoms and the changes in x, y and z direction caused by this, a MR image is generated. Then, the detected signal is transformed by Fourier Transform, amplified, and presented in a computer. MR images could be acquired with different sets of pulse sequences and gradients which results in different image appearance. In this assay, after finishing the 3D spheroid growth, MRI studies were performed to validate the in vitro T2 contrast capability of mSLNs for MRI applications. Thus, a change in relaxivity was analyzed after the internalization of mSLNs in Hs578t polyspheroids. MR imaging was performed in a MR Solutions Benchtop scanner (Guildford, UK), under a clinical magnetic field of 3.0 T horizontal bore, at room temperature. To measure the samples, polyspheroids were placed in a 200 µL of Milli-Q water in a Petri dish (each compartment for each condition). The plate was set into a custom printed MRI holder and positioned in the center of the MRI scanner bore. Subsequently, a imaging acquisition took place using a fast spin-echo (FSE) sequence, with parameters mentioned in Table V. Post-processing was completed using the ImageJ software (1.46r, NIH, USA), to determine the relaxation time for 41 each condition, using the “MRI analysis calculator” plugin. To calculate the transverse relaxivity (r2) of the mSLNs, the Equation 6 was used. 𝑟2=1/𝑇2 Equation 6 Table V. Parameter of the fast spin-echo (FSE) sequence used for imaging acquisition. Using the fast spin-echo (FSE) sequence, the relaxation time for each condition was acquired with the parameters shown in this table. Parameters Values Matrix 4096 x 2048 Field of view (FOV) 40 x 40 mm Slices 6 Slice thickness 1.5 mm Echo time (TE) 15 ms Repetition time (TR) 1500 ms Number of averages (NA) 5 Total acquisition time (AT) 581 ms 4.4. CONFOCAL MICROSCOPY IMAGING The confocal microscope LSM780 from Zeiss was used to visualize compound internalization into Hs578t spheroids. After spheroid formation, one spheroid/well/500 µL were placed in a Petri dish divided in 4 compartments. The spheroids were treated with 10 µM of respective compounds, and then incubated overnight at 37 ºC under 5% of CO2. Thereafter, spheroids were washed with 1x PBS and Hoechst 33342 was added, at a concentration of 0.1% of total volume. The spheroids were incubated for 10 min and visualized by confocal microscopy. The inherent fluorophore (DiO) incorporated in mSLNs membranes permitted to track the designed nanoparticles, and additionally, the DOX inherent fluorescence was followed and co-localized with a nuclear stain (Hoechst 33342), using the excitation laser of 488 nm for DOX and DiO, and 405 nm for Hoechst 33342. 4.5. INDUCTIVELY COUPLED PLASMA-OPTICAL EMISSION SPECTROSCOPY Inductively coupled plasma-optical emission spectroscopy (ICP-OES) is a broadly used analytical technique to determinate and quantify trace elements in complex sample formats (0.1 – 100 ng.mL-1) (210,211). ICP-OES can simultaneously detect up to 70 elements, being widely used in nanomedicine to quantify the percentage of specific elements ( e.g., Fe) in nanoparticles. This 48 3| RESULTS AND DISCUSSION 49 1. DOSE RESPONSE CURVES AND IC50 DETERMINATION The therapeutic effect of the developed mSLNs probes (Table IV) was studied by determining the IC50 (concentration that inhibits 50% of cell viability) using the resazurin assay. Hs578t and MDA-MB-231 cancer cell lines were treated with 14 different concentrations of different mSLNs: 100, 50, 25, 12.5, 5, 2.5 1.25, 0.63, 0.31, 0.17, 0.08, 0.04, 0.02, and 0.01 µM relative to DOX content, without an external magnetic stimulus, for 24, 48 and 72 h of incubation. DOX, a cytotoxic drug used in breast cancer treatment, was used as reference compound to compare the cytotoxic effect of this commercial drug with DOX-loaded mSLNs. The results from the IC50 determinations for both cell lines are summarized in Table VI and the dose response curves are represented in Figure 13 and Figure 14 for Hs578t and MDA-MB-231 cell lines, respectively. Table VI. IC50 values of free DOX and DOX-loaded mSLNs for Hs578t and MDA-MB-231 TNBC cell lines. The IC50 values were determined for three different times of incubation, 24, 48 and 72 h. Compound[a] Hs578t MDA-MB-231 24 h IC50 (µM) ± SD[b] 48 h IC50 (µM) ± SD[b] 72 h IC50 (µM) ± SD[b] 24 h IC50 (µM) ± SD[b] 48 h IC50 (µM) ± SD[b] 72 h IC50 (µM) ± SD[b] DOX 8.88 ± 2.20 4.47 ± 0.997 2.26 ± 0.29 12.21 ± 1.258 3.40 ± 0.19 3.06 ± 0.25 EBR52 3.55 ± 0.64 3.31 ± 0.39 2.27 ± 0.54 3.75 ± 0.21 3.10 ± 0.22 2.58 ± 0.51 EBR85 3.73 ± 0.40 2.35 ± 1.08 1.82 ± 0.60 2.98 ± 0.24 1.35 ± 0.06 1.13 ± 0.19 EBR86 3.49 ± 0.35 2.29 ± 0.64 1.98 ± 0.43 2.28 ± 0.14 1.74 ± 0.51 1.23 ± 0.19 EBR89 n.d. n.d. n.d. n.d. n.d. n.d. [a]: Each treatment was tested, at least, in triplicate and the date are presented as mean values. [b]: Standard Deviation. n.d.: non-determined 50 Figure 13. Dose response curves and IC50 comparisons. Effect of mSLNs (EBR52, EBR85, EBR86, and EBR89) and reference compound (free DOX) on the cell viability of Hs578t cancer cells, for total cell biomass, at 24, 48, and 72 h of incubation. Results are expressed as a mean ± SD. a : Results significantly different from EBR52, EBR85, EBR86, and EBR89 ( p <0.0001); b : Results significantly different from DOX, EBR52, EBR85, and EBR86 ( p <0.0001); c: Results significantly different from EBR85, and EBR86 ( p <0.05). 51 Figure 14. Dose response curves and IC50 comparisons. Effect of mSLNs (EBR52, EBR85, EBR86, and EBR89) and reference compound (free DOX) on the cell viability MDA-MB-231 cancer cells, for total cell biomass, at 24, 48, and 72 h of incubation. Results are expressed as a mean ± SD. a : Results significantly different from EBR52, EBR85, EBR86, and EBR89 ( p <0.05); b: Results significantly different from DOX, EBR85, EBR86, and EBR89 ( p <0.05); c: Results significantly different from DOX, EBR52, EBR85, and EBR86 ( p <0.0001); d : Results significantly different from DOX, EBR85, and EBR86 ( p <0.0001) 52 The functionalized nanoparticles (EBR85 and EBR86) were the most active molecules, in both cell lines, with a capacity to reduce the cell viability in a dose-dependent manner and displayed the lowest IC50 value at 72h of incubation, in comparison with DOX-treated cells, which could be indicating a sustained drug release along time. Additionally, it was possible to observe that the nanoformulations without drug (EBR89) are non-toxic for cancer cells within the whole range of drug concentrations tested. For a safe biological application of nanocarriers in human tissues, their toxicity should be lower or negligible. The resazurin fluorometric assay demonstrated that EBR89-functionalized nanoparticle presented a good biocompatibility at the highest concentration tested (0.014 mgFe.ml-1). According to the literature, these results are consistent with other reported results, revealing only cytotoxicity effects for higher Fe concentrations (219,220). The synergistic toxicity of chemotherapy in combination magnetic hyperthermia combination was also assessed using the resazurin assay, in Hs578t cells. The objective of this dual treatment was to increase the therapeutic index of chemotherapeutic treatment alone, thus allowing the use of reduced drug doses and avoiding the harmful side effects associated to standard chemotherapy treatments. Therefore, Hs578t cells were treated with 6 different concentrations, 0.1, 0.3, 0.6, 1.2, 2 and 5 µM related to DOX content, followed by 1 h of an external magnetic stimulus application and 72 h of incubation. The dose response curve and the graph representation of the IC50 values obtained with or without magnetic hyperthermia application are presented in Figure 15 and the IC50 values are summarized on Table VII. Additionally, using the IC50 values obtained for the cells-treated with or without magnetic hyperthermia application, the activity gain of each formulation was calculated for the dual treatment using the Equations 1 and 2 (Chapter 2, section 3). 53 Table VII. IC50 values of the mSLNs and Doxorubicin for Hs578t cancer cell line. The IC50 values were determined for 72 h of incubation after treat the cells with respective compound plus 1 h of hyperthermia. Treatment[a] IC50 (µM) ± SD[b] Only Hs578t cells + HT n.d. DOX + HT 2.1 ± 0.11 EBR52 + HT 0.62 ± 0.0.7 EBR85 + HT 0.21 ± 0.16 EBR86 + HT 0.60 ± 0.02 EBR89 + HT 1.84 ± 0.26 [a]: Each treatment was tested, at least, in triplicate and the date are presented as mean values. [b]: Standard derivation. HT: Hyperthermia application. n.d.: non-determined. Figure 15. Cytotoxic effects of different mSLNs on Hs578t cells. A. Effect of mSLNs (EBR52, EBR85, EBR86, and EBR89) and reference compound (free DOX) plus 1 h of hyperthermia on Hs578t cancer cells, for total cell biomass, after 72 h from treatment. B. Comparison of the IC50 values obtained for the cells-treated with and without the application of a magnetic stimuli. Results are expressed as a mean ± SD. 54 The results demonstrated that the main difference between the formulations can be observed under dual-treatment application to the cancer cells, with significant cancer cell viability decrease. EBR89 formulation displayed the highest activity gain and, otherwise, the highest IC50 value, in comparison to the other nanoparticles (Figure 15 (B)). This is an expected result, as this formulation does not contain DOX. MH effect is inherent to the presence of magnetite inside of the nanoparticles, where its activity as nanoheaters is controlled by applying an AMF (221), being considered the MH itself as an anticancer promoter (222). Indeed, the increased activity observed on EBR89-treated cells is intrinsic to the MH activity. However, the major anticancer activity is observed under thermo-chemotherapy treatment, where the EBR85-functionalized nanoparticle displayed the lowest IC50 value (0.212 µM) with a highest percentage of activity gain among the DOX-containing formulations (>80%), in Hs578t cells, indicating a strong potential for its application as a combinatorial treatment. The results obtained in the presence of an AMF showed that the EBR85-functionalized nanoparticles are effectively transporting and delivering the drug and promoting a cell viability decrease compared to the experimental condition in which no hyperthermia was applied (0.21 ± 0.16 µM vs 1.82 ± 0.60 µM). Microscopically, the effect of dual thermo-chemotherapy versus monotherapy (chemotherapy alone) was observed by morphological alterations in the Hs578t cells, after 72 h of incubation after treatments, with and without MH, using the respective IC50 DOX concentration of the different mSLNs (Table VII) (Figure 16). The combined thermo-chemotherapy treatment induced cell phenotypic alterations and apparent cell death, as observed in Figure 16 (C). Chemotherapy is associated with adverse side effects for the patients, such as cardiotoxicity in the case of DOX treatments (64,76), being urgent to discover new strategies to overcome these Figure 16. Morphological alterations in Hs578t cancer cells after different treatment typology. Phenotype of Hs578t cancer cells treated with only medium (A), EBR85 at respective IC50 concentration (B) , and with EBR85 at respective IC50 concentration and 1 h of MH application (C), at 100X of magnification. A C B 55 issues. One possibility would be increasing the therapeutic index of the treatment in such a way that lower chemotherapeutic doses could be administered to the patient, thus preserving normal tissues from damage. The synergistic anticancer effect of the thermo-chemotherapy treatment was determined by the analysis of the corresponding synergistic degree (Table VIII) calculated from Equation 3 (Chapter 2, section 3). As illustrated in Table VIII, interactions between EBR85functionalized nanoparticles and hyperthermia showed the highest value of synergistic effect for the Hs578t cancer cell line, a result already observed in Figures 15 and 16. This indicates that the dual treatment using the novel functionalized mSLNs probes enhance the anticancer therapeutic index face to chemotherapy or MH alone, indicating a possible breakthrough for future therapies on TNBC with a possible reduction of conventional chemotherapy side effects. Table VIII. Degrees of synergism for different therapy combination. Alpha (α) values calculated with Equation 3 (Chapter 2, Section 2) for different mSLNs formulations in combination with an alternating magnetic field. Treatment α Result DOX 1.08 Addictive effect EBR52 3.66 Synergistic effect EBR85 8.67 Synergistic effect EBR86 3.30 Synergistic effect 2. EFFECT OF NANOPARTICLE SURFACE FUNCTIONALIZATION ON IMMUNE SYSTEM RESPONSE In the systemic administration of nanoparticle-based treatments, the opsonization process starts when the nanoparticles are injected in the bloodstream, initiating a rapid MPS action. As a result, the clearance of the nanocarrier from the circulation occurs within a few hours post-injection. Herein, the biophysiological functionality of the nanocarrier is achieved through the mSLNs surface functionalization with a temperature-responsive biocompatible hydrophilic polymer (PEG)- containing ligand. This specific surface modification resists to the immune cell interaction, avoiding opsonization and, consequently, its elimination from the blood circulation (172,173). In addition, 56 to avoid the immune response, PEGylation enhances the probability of its passive accumulation in the tumor region. THP-1 cell line was used as model for monocyte cells which are involved in the innate immune response, responsible to recognize and create a response against foreign pathogens (223). In order to study the response of monocytes cells against nanoparticles, a cell viability assay was conducted using the resazurin colorimetric assay. THP-1 cells were treated with 6 different concentrations using a range of concentrations 0.1, 0.3, 0.6, 1.2, 2 and 5 µM related to doxorubicin content, without hyperthermia application, for 24, 48 and 72 h of incubation. The dose response curve and the graphic representation of the IC50 values obtained are illustrated in Figure 17 and summarized in Table IX. 57 Figure 17. Dose response curves and IC50 comparisons. Effect of mSLNs (EBR52, EBR85, EBR86, and EBR89) and reference compound (free DOX) on the cell viability of THP-1 cells, for total cell biomass, at 24, 48, and 72 h of incubation. Results are expressed as a mean ± SD. a : Results significantly different from EBR85, EBR86, and EBR89 ( p <0.05); b: Results significantly different from DOX, EBR85, EBR86, and EBR89 ( p <0.05); b : Results significantly different from DOX, EBR52, EBR85, and EBR86 ( p <0.0001) 64 agreement with the literature where it is described a sustained drug release along time from the nanoparticles, thwarting the “burst effect”, contrary to an immediate exposure to a total DOX concentration (84,93,94,97,125,141,142). With an increase in incubation time (6h post-treatment), the cell nuclei exhibit a higher DOXred fluorescence, principally on EBR85-treated cells. After nanoparticles were internalized into Hs578t cancer cells, a continuous DOX liberation from the nanoparticle formulations was promoted where, it was first released into the cell cytoplasm and with increasing incubation time, could reach the cell nuclei and consequently induce cancer cell death. 6. EFFECT ON 3D HS578T POLYSPHEROID GROWTH The monolayer cell culture (or 2D culture) is a method often used in the study of cellular responses to drug testing, pre-clinical in vitro assays, including for cancer research. The high reproducibility, low cost, simplicity, and of this well-established model enhances its attractivity for in vitro studies, being the most commonly used method for cell culture. Though, different studies pointed out large discrepancies between in vitro and in vivo results, highlighting the difficulty to replicate the complex tumor microenvironment and the drug resistance observed in solid tumors (208,209). 3D culture models have come to fill the gap between in vitro and in vivo systems. Spheroids are conjugation of cell aggregates in the order of micro-sized, generated from a cancer cell line that have emerged as a new tool for cancer research. Different drug studies have used spheroids as models for different cancer types, such as breast, colon, lung, pancreas, liver (226,227). The drug resistance detected on solid tumors is inherent to the failure of subscribed chemotherapies. Due to cell agglomeration established on 3D tumor spheroids, the physical communication and signaling pathways could be mimicked, a feature that plays a role in the chemotherapy resistance observed in solid tumors (226). Within the cells’ agglomerate, different ECM constituents (e.g., collagen laminin, fibronectin) are deposited, supporting ECM-cell interactions which are responsible for a barrier formation that limits the penetration, distribution, and action of anticancer drugs (227). Additionally, as occurs in solid tumors, 3D tumor spheroids are divided into different layers responsible to impair the therapeutic efficacy of anticancer drugs (228). The external layer is constituted by highly proliferative cells, while the middle contains quiescent cells and the inner core is formed by necrotic cells, where the location of the cells is correlated to their capacity to capture nutrients and oxygen (Figure 23) (226–228). 65 Different reports demonstrated different outcomes for drug uptake and action in 2D cell layers and 3D spheroids (226,227). Taking this into consideration, the mSLNs effect on 3D tumor growth was evaluated and compared to the 2D monolayer results. Hs578t polyspheroids were treated with 2xIC50 concentration of the developed nanoparticles (EBR52, EBR85, EBR86, and EBR89) and reference compound (DOX) plus 1 h of AMF every second day for 1 week (Figure 24 (A)). Then, tumor growth was assessed during the following 2 weeks with photographic registration. The normalized polyspheroid growth was obtained using the Equation 4 (Chapter 2, section 4) and plotted on Figure 24 (B). Figure 23 3D spheroids schematic representation. Spheroids are divided into three main layers (proliferation zone, quiescent zone, and necrotic zone), caused by a gradient of oxygen, nutrients, pH, CO2, and metabolic waste, similar to what is observed in solid tumors. 66 Polyspheroids treated with a combination of reference compound (DOX) plus 1h of MH displayed an inhibitory growth effect until day 8 of the experiment, but over time, the spheroids reinitiate an exponential growth (Figure 24 (B)). Previous reports already demonstrated that breast cancer cell lines cultured in 3D conditions showed more chemotherapeutic resistance in Figure 24. Effect of dual-treatment on polyspheroids growth. (A) Chronological representation of the polyspheroids treatment for three weeks . (B) Graphical representation of the spheroid tumor growth after treated with 2xIC50 concentration of respective compound plus 1 h of hyperthermia for three days in one week. Results are presented as mean ± SD of at least three independent experiments . (C) Graphical representation of normalized polyspheroids daily growth after performed a treatment 3 times per week with respective compounds and 1 h of hyperthermia. Results are presented as mean ± SD of at least three independent experiments . a: DOX and EBR52 significantly different from EBR85, EBR86, and EBR89 ( p <0.0001); b: EBR85 significantly different from DOX, EBR52, EBR86, and EBR89 ( p <0.0001); c: EBR86 significantly different from DOX, EBR52, EBR85, and EBR89 (p<0.0001); d: EBR89 significantly different from DOX, EBR52, EBR85, and EBR86 ( p <0.0001). 67 comparison to those cells in 2D conditions (79). Similarly, EBR52-treated cells displayed the same growth behavior as DOX-treated cells, highlighting the key role of the surface modifications on the sustained drug release that enables a more prolonged treatment. In agreement with 2D cell monolayer results, EBR89-treatment seems to exhibit the least effect on Hs578t spheroid growth. This means that the monotherapy application (only hyperthermia) did not display a cytotoxic effect on cancer cells, enhancing the reliability of their use for diagnostic proposes. The functionalized nanoparticles, EBR85 and EBR86, were the nanoparticles with the best growth inhibitory capacity. For a better visual appreciation, the daily growth of the tumors were plotted on Figure 24 (C). Positive values represent the positive growth of the polyspheroids, where the compounds did not demonstrate a therapeutic effect, whereas negative values correspond to the spheroids which were susceptible to the treatment. As previously mentioned, spheroids can reproduce the mechanisms of drug resistance observed in solid tumors, being a feasible methodology for the study of new therapeutic approaches. In Figure 24 (C), it was shown that functionalized mSLNs-treatment potentiates the effect of the chemotherapeutic drug along time with a higher inhibition on tumor daily growth, in comparison to DOX-treatment alone, enhancing the cell sensitivity to chemotherapeutic treatment, as previously dated (79). As observed in Figure 24 (C), the functionalized mSLNs displayed the best inhibitory effect on polyspheroids daily growth, where the EBR86-treated group showed a higher inhibition on polyspheroid tumor growth in comparison to EBR85-treated group. The application of an AMF is supposed to selectively activate the magnetite of the nanoformulations (Figure 25). This generates energy in the form of heat that consequently breaks the thermo-susceptible Diels-Alder bond, exposing the penetrating peptide to the cancer cells (177,181,183). Then, the nanoparticles could be easier and faster internalized into cancer cells, accelerating the delivery of the chemotherapeutic drug and also of the magnetite nanoparticles (used as a contrast agent for MRI) to Hs578t polyspheroids. Once inside, the cells will degrade the magnetic solid lipid nanoparticles to liberate the chemoand thermo-deadly cargo. This will damage the affected cells and diminish their proliferation rates (180,182). However, the effect of the temperature-responsive Diels-Alder bond contained in the PEGylated ligand on the spheroid proliferation rate was similar to that of the temperature-insensitive control formulation, what points to an effective spheroid penetration of the 68 mSLNs even when the targeting mechanism trough the CPP is not thermally activated (Figure 24 (C)). On the other hand, MH action is intrinsically dependent on the concentration of magnetite nanoparticles inside the mSLNs, in such a way that a higher concentration of magnetite is correlated to a higher generation of heat. In this way, the concentration of magnetite inside the different mSLNs could be behind the previous findings regarding the tumor growth. Since low magnetite concentrations inside the mSLNs functionalized with the temperature-sensitive PEGylated ligand could not be enough heat as to be able to activate the CPP-mediated targeting effect, what would justify the non-improved spheroid growth inhibition observed for EBR85 particles. To clarify this issue, and to study the nanoparticle internalization into TNBC Hs578t cells, Fe content was quantified by ICP-OES at the end of the polyspheroid growth rate experiments (Figure 26). Figure 25. Diels-Alder bond after hyperthermia application. The hyperthermia application will activate the magnetite that, consequently, will generate an enough amount of heat to break the thermo-susceptible diels-alder bond. Afterwards, the cell-penetrating peptide ligand will be exposed to the cancer cells, facilitating the nanoparticle cell internalization. 69 As observed in Figure 26, the amount of Fe was measured in the DOX-treated polyspheroid. The values obtained, named as real values, correspond to the amount of Fe present in cells and medium. Once this value was subtracted to the real values achieved in each treated polyspheroid group, the resulting Fe content accounts for that actually internalized together with the mSLNs nanoformulations. Lastly, Figure 27 compares the measured Fe content coming from the mSLNs spheroid cell internalization to the actual Fe content coming from the mSLNs nanoformulation dispersions. Figure 26. Fe internalization in Hs578t polyspheroids after treatment with compounds plus 1 h of MH. At the end of the polyspheroid growth rate experiments, Fe (magnetite) internalization into polyspheroids was assessed by quantitative analysis of Fe through ICP-OES. Results are presented as mean ± SD of at least three independent experiments. Figure 27. Correlation between the Fe concentration of the stock nanoformulations and the Fe content amount internalized into the polyspheroid cancer cells. (A) Fe concentration of stock solutions versus Fe content of polyspheroid cancer cells, and (B) linear regression of the previous plotted results. The linear regression has the follow equation: µg Fe = 0.011 [Fe of nanoformulation stock] + 0.057 with 0.929 value for r2. 70 On Figure 27 (A) and (B), a clear strong relationship between the Fe concentration inside of mSLNs dispersions and the Fe content internalized into the polyspheroids is appreciated. These parameters show a linear and positive dependency. As a final outcome, it was observed that EBR85-treated polyspheroids had a lower Fe content, in comparison to EBR86-treated cells, which could be a possible explanation for the unexpected results regarding the lower polyspheroid growth rate observed for EBR85-treated polyspheroid. Following this, the polyspheroid growth rate was evaluated taking into consideration the quantified Fe content inside the polyspheroid cancer cells (Figure 28). Using ICP-OES, it was possible to determine the Fe content internalized into polyspheroids study its effect on polyspheroids’ growth rate. As observed in Figure 28, the EBR85-treated group displayed a significant inhibition on tumor daily growth in comparison to EBR86-treated group with the same amount of Fe internalized. When considering the internalized magnetite, the effect of the Diels-Alder-mediated targeting activation was evidenced, which enhances the nanoformulation activity against cancer cells. This effect seems to be Fe dose-dependent, where a higher Fe content potentiates the inhibitory effect at the tumor growth level, an outcome already observed by Hayashi et al. where they demonstrated that an enhanced accumulation of nanoparticles increases the magnetic relaxivity (229). Altogether, EBR85-functionalized mSLNs induced a higher inhibitory effect on tumor growth in comparison to the other mSLNs formulations, when the effect its normalized to the amount of Fe internalized, thus overcoming a possible drug resistance observed Figure 28. Effect of compounds on tumor daily growth rate considering the Fe content internalized into polyspheroid cancer cells. Graphical representation of normalized polyspheroids daily growth per mg/ml of internalized Fe. Results are presented as mean ± SD of at least three independent experiments. * p <0.05 71 for patients treated with chemotherapy and maximizing the DOX action on tumor cells. These results reinforce the potential use of EBR85 as a new therapeutic approach for TNBC treatment faced to conventional chemotherapy. 7. FUNCTIONALIZED NANOPARTICLES AS T2-CONTRAST AGENTS FOR MRI The MRI technique is employed for a rapid in vivo image; however, the associated low sensitivity challenges its use for diagnostic proposes, which appeals for the use of magnetic CAs overcome this issue (118). Fe3O4 nanoparticles made it into the clinical setting as T2 CAs, with associated limitations regarding the magnetic susceptibility artifacts which may not clearly differentiate the higher from the lower of MRI signal levels arising from adjacent tissues (230). In this context, the efficacy of the developed mSLNs was evaluated in terms of MRI performance as T2 CA. The developed nanoformulations have the nanosized magnetite incorporated in the organic matrix, so T2-MRI behavior (dark contrast enhancement) is expected (85). In this way, the magneticallylabelled polyspheroids used in the previous experiment were utilized to study the MRI contrast enhancement effect of the mSLNs in comparison to DOX-treated cells, using a preclinical 3 T MRI scanner under T2 acquisition modes using fast spin-echo (FSE) sequences (Figure 29). 72 Surface functionalization acts as an improvement for nanoparticle internalization into cancer cells, where its fault will significantly diminish the endosomal internalization of nanoparticles and, consequently, decrease intracellular magnetite content. A T2-MRI behavior is correlated to a long range of magnetic interaction with water molecules induced by the superparamagnetic core under a magnetic field, which results in hyperintense changes in the resonance signal in MRI imaging (85,113,115). Thus, the MRI contrast enhancement is a concentration dependent phenomenon, Figure 29. MRI contrast enhancement effect of magnetically-labelled polyspheroid cells. In (A) images obtained using preclinical 3 T MRI scanner under T1 and T2 acquisition modes using fast spin-echo (FSE) sequences for the treated polyspheroids. Using the ImageJ with MRI-T2 calculation plugin, it was obtained the graphic represented in (B) for the T2 values (s, at 3 T 37 ºC) of the different treatments applied. Results are presented as mean ± SD of at least three independent experiments. Statistical analysis using the ANOVA tests where * p <0.05, *** p <0.001,**** p <0.0001 in comparison to the other compounds-treated polyspheroids. 73 the higher the concentration of magnetic nanoparticles, the lower the relaxation time, and the darker MRI contrast effect, as observed in Figure 29. The functionalized nanoparticles EBR85, EBR86, and EBBR89 (all containing magnetite nanoparticles in the organic matrix) showed an enhancement of the T2-MRI contrast in comparison to the other nanoformulations with EBR85 probe displaying the lowest T2 value, without significant differences among the treatments (Figure 29). As previously observed, EBR85-treated polyspheroids had lower magnetite, meaning that magnetic dipolar interactions and related clustering effects coming from the confinement of the MNPs in the wax matrices could be playing a major role in the final relaxation times observed, as already demonstrated in other reports (231). Additionally, it is important to highlight that the transverse relaxivity r2 – figure of merit that quantifies the efficiency of MNPs as CAs – calculated as the slope of the inverse of the relaxation time (T2 values from Figure 29, Equation 6 from chapter 2, section 4) versus the concentration of the active magnetic element of the functionalized-EBR85 mSLNs was of 120 mM-1s-1, value that outweighs that of Feridex® (r2 = 93 mM-1s-1, at 3 T, 37 ºC), superparamagnetic iron oxide nanoparticle already approved by FDA as negative CAs (188). Altogether, EBR85 showed a remarkable effect on the reduction of the T2-MRI relaxation time, becoming promising as T2-MRI contrast agent able to offer a noninvasive imaging capability for diagnosis and monitorization during cancer treatment. 8. EFFECT OF THE NANOPARTICLES ON INVASION CAPACITY OF HS578T SPHEROIDS A malignant tumor is characterized for its potential to invade surrounding tissues and spread to distant organs, being responsible for the majority of the cancer-related deaths or related to higher probability of cancer relapse within a period of 5-years (2,19). This brings a new perception to the validation of novel therapies. The implementation of novel anticancer drugs should pass through an in vitro test that allows the identification of an inhibitory capability to this key hallmark of cancer. A 3D tumor spheroid invasion assay was performed as a novel approach, being a rapid and highly reproducible technique that has an associated capacity to reproduce a tumor region with ability to create micro-metastasis (232,233). This task started with the development of single Hs578t spheroids and, 4 days post-development, the invasion assay was initiated. Matrigel was added directly to each well to provide a semi-solid matrix which enabled the invasion of cancer 80 In published studies, Hs578t cells were used for ex vivo angiogenesis studies to evaluate the effect of the functionalized zeolites as proangiogenic or antiangiogenic agents. It was demonstrated that the functionalized zeolites decreased the recruitment and formation of blood vessels to the tumor region face to non-functionalized probes (236). The poor outcome and the presence of higher levels of angiogenesis have been associated with different cancer types, as for breast cancer, renal cancer, prostate cancer, colon cancer and melanoma (237). Thus, there is a need to validate the novel therapies for their ability to inhibit or decrease angiogenesis and, consequently, tumor progression. After 4 days of tumor development, the formation of blood vessels is noticeable on the control group (as observed in Figure 33 (A) and in Figure 34), whereas the treated groups showed a significant decrease in the number of blood vessels, in comparison to the control. Although EBR85treatment induced a higher decrease in angiogenesis in comparison to DOX, suggesting an improvement in blood vessel formation, this difference was not significant ( p >0.05). For further characterization of the nanoparticles’ anticancer activity, Ki67 immunohistochemistry analysis were performed. Ki67 protein is involved in all cell cycle phases (G1, S, G2, and M), with an absent expression during the cell resting phase (G0) (238). The expression of this protein is strongly correlated with active cell proliferation of intrinsic cell populations in tumor cells, allowing its use as a biomarker of tumor aggressiveness. In invasive breast cancers, Ki67 biomarker is strongly applied to stratify good and poor prognostic Figure 34. Effect of nanoparticles EBR85 and DOX on Hs578t tumor angiogenesis. Graphical representation of the number of blood vessels formation in response to a different treatment. Data was analyzed by one-way ANOVA and the results are representative for the mean of at least 15 eggs per group;*** p <0.0001 * p <0.05 in comparison to the control group. 81 classifications (16,22). The proliferation status of breast cancers is also a guiding decision for the cancer response to the prescribed chemotherapy where low-proliferative tumors are less sensitive to such therapies. In this study, Ki67 expression analysis was performed on sections from formalinfixed, paraffin-embedded CAM cancer samples. Scoring was assigned as 0 to 0% of positive cells, 1 to <10% of positive cells, 2 to 10-50% of positive cells and 3 classifications for >50% of positive cells. (Figure 35). Ki67 scores are used in the clinics as biomarker for the response to chemotherapy in TNBC (239). A high proliferative index was detected by Ki67 immunostaining in the control-treated group, where a large number of brown cells was detected, which usually correlates to TNBC aggressiveness. A study with 613 breast carcinoma samples was conducted and followed-up for 10 years, being observed that a Ki67 high expression was correlated to a significantly higher risk Figure 35.. Effect of EBR85 and DOX on angiogenesis of Hs578t cancer cell line ex vivo , detected by chick chorioallantoic membrane (CAM) assay at 96 h post-treatment. In (A) representative images of the excised CAM tissue sections of the control, treated (DOX and EBR85) conditions, immunostained for Ki67. Positive immunoreactions for Ki67 in malignant cells are shown in brown (DAB staining). Original magnification at 100x and 200x (insets). (B) is a graphic representation of the attributed scores for each section cuts, where 0 was assigned to 0% of positive cells, 1 to <10% of positive cells, 2 to 10-50% of positive cells and 3 classifications for >50% of positive cells. Results are expressed as the mean ± SEM of one experiment. * p <0.05 relative to control. 82 of shorter tumor-free patient survival (240). These findings highlight the importance to discover new drugs that have cell anti-proliferative effects to improve TNBC prognosis. The functionalized nanoparticle EBR85 showed promising results at this level, revealing a significant reduction in tumor cell proliferation in comparison to control group (0.3±0.5 versus 1.25±.1.0, p <0.05). Ki67 quantification for DOX-treatment revealed a higher proliferative activity, demonstrating its lower effectiveness compared to EBR85 mSLNs. Altogether, these initial results suggest that EB85treatment induces an important reduction in cancer cell proliferation in TNBC. 11. IN VIVO THERANOSTIC EFFECT OF FUNTIONALIZED NANOPARTICLES USING ORTHOTOPIC TNBC XENOGRAFTS Part I - Toxicity and diagnosis Based on the promising in vitro properties of the DA-TAT-PEG-mSLNs (EBR89), which revealed no toxicity for both 2D monolayer cells, 3D Hs578t spheroids, and ex vivo CAM model, the developed nanoprobes were evaluated in vivo on Hs578t tumor bearing immunocompromised female NSG mice. The mice were inoculated orthotopically with Hs578t cells (650 000 cells per mice) in a mammary fat pad. Treatment started 3 days post-tumor implantation and mice were randomly divided into three groups. Mice-bearing Hs578t tumors were injected intravenously every 2 days for 1 week with: Treatment 1 (saline solution), Treatment 2 (EBR89 at 0.05 mgFe.Kg-1 of mice weight), and Treatment 3 (EBR89 at 0.05 mgFe.Kg-1 of mice weight plus 1 h of an AMF using the following settings: 15.4 A of current, 15 V of voltage, and 181 kHz of frequency. Tumor progression was evaluated once a week after finishing the treatment (Figure 11, Chapter 2, section 6) The animal survival rate was 100% with all animals reaching the predetermined 43-day endpoint. 83 Treatment with EBR89 did not display any difference in tumor growth in comparison to the control group, indicating that EBR89 particles do not promote any anticancer activity (Figure 36 (A)). The same outcome was observed for the group which benefited from the hyperthermia application (EBR89 + HT), where there was no significant tumor growth inhibition effect compared to the other groups ( p >0.05). This goes in agreement with the in vitro results where it was demonstrated that EBR89 does not display different complementary mechanisms to promote cancer cell selective toxicity, even in the presence of an external magnetic stimuli. Additionally, mice body weight was monitored to assess the overall effect of the tumors and treatment on the animals (Figure 36 (B)). No significant body weight loss was observed during the experimental period in neither group. On sacrifice day, the tumors were collected from the animals and were weighed. As observed on Figure 36 (C), no differences were observed between the treatments, supporting the inefficacy of these nanoformulations in vivo . Altogether, the developed nanoparticles themselves appear to be safe vehicles in systemic circulation, not affecting the cancer cell growth, which support their application in early diagnosis. Figure 36. Evaluation of nanoparticle toxicity on mice-bearing Hs578t tumors. The 3 groups of mice were treated with saline solution, EBR89, or EBR89 plus 1 h of hyperthermia, three times for one week and humanely euthanized 35 days post-tumor implantation. ( A) effect of the treatment on tumor growth along time, (B) graphical representation of the body weight of the animals, and (C) weight of tumors collected 43 days post-implantation. 84 The efficacy of the nanoformulations to specifically target the cancer cells and provide a MRI contrast enhancement in vivo was evaluated post-mortem in NSG mice bearing Hs578t xenograft tumors. The specificity of the nanoprobes was evaluated by comparing the contrast enhancement of the tumor regions of mice receiving the combination of EBR89 with an AMF and those receiving only the EBR89 nanoparticle at the same concentration, as well as comparing the contrast enhancement with the group which received the vehicle. In vivo T2*-weighted MRI images (Figure 37) were taken 43 days post-tumor implantation (30 days after the last treatment day (Figure 11, Chapter 2, section 6). Figure 37. Post-mortem T2*-weighted MRI images taken from a NSG mice bearing Hs578t tumors. (A) On the top, are the representative in vivo T2 MRI images obtained from a preclinical 3 T MRI scanner under T1 and T2 acquisition using fast spin-echo (FSE) sequences and, at the bottom, are the color-mapped images obtained from MRIT2 calculation plugin of ImageJ program, 30 days after intravenous injection of EBR89 nanoparticles, EBR89 followed by hyperthermia (HT), or only vehicle into a NSG mice bearing Hs578t cells (650 000 cell injection). The red arrows define the tumor location in the different representative MR images and the green ones demonstrate the same location on treated images (through ImageJ). The color-scale was obtained through ImageJ. The graphic represented in (B) was obtained using the ImageJ with MRI-T2 calculation plugin for the T2 values (s, at 3 T 37 ºC) concerning the different treatments applied. Results are presented as mean ± SD. Statistical analysis using the ANOVA tests where * p <0.05, *** p <0.001,**** p <0.0001 in comparison to vehicle. 85 Different articles assessed the use of iron-based nanoparticles in vivo as MRI contrast agents. The usual procedure involves the evaluation of in vivo T2-weighted images of mice-bearing tumor models at 0, 2, 4, 8, 24, and 48 h post-nanoparticles injection (reviewed in (241)). Only a few studies focused on tumor detection by MR imaging at a longer time post-injection, being 14 days the maximum studied time, where the nanoparticles could still be detected in the tumor region (242,243). The focus of this part of the in vivo studies was to i) assess the nanoparticle toxicity; and ii) evaluate, post-mortem, the nanoparticles’ efficacy as T2-contrast agents for diagnostic applications and monitorization of tumor response to therapy. As observed in Figure 37, the functionalized nanoparticles without drug - EBR89 - were able to significantly diminish the T2 value in comparison to the control group, demonstrating its efficacy as T2-contrast agent for MRI application. A better T2 profile was observed in the presence of hyperthermia application. These results can be correlated to the activation of Diels-Alder bonds which would trigger enhance the nanoparticle internalization into cancer cells. To improve the MRI final image, a higher concentration of nanoparticles inside the cells is required, which will promote a superior dark contrast. In a published study, the authors pointed to 60 mgFe.Kg-1 of mice as the best iron concentration to get good quality images, being considerably superior to the concentration used here (13.5 mgFe.Kg-1 as accumulative dose) (242). Though, the concentration tested here seems to be enough for MRI application. Altogether, these results are in agreement with those observed in vitro , where the influence of the surface activation by an external stimulus was observed. This confirms that, these new nanoparticles have the potential to act as probes in early tumor diagnosis. At the end of the experiment, heart, lungs, kidneys, liver, and tumor were collected from each animal for further chemical analysis. Histological slices from the collected organs of the control and treated animals were stained with hematoxylin and eosin (H&E staining). As observed in Figure 38, no organ damage was observed in terms of histology, in comparison to the control group, supporting a safe profile. Though, an analytical confirmation is still required. Liver and kidney function is correlated to physiological toxicity, where the evaluation of liver enzymes could be used as a key biomarker to understand the extension of liver cell damage (87). So, in future work, it will be interesting to know the creatinine values in the animal serum. 86 Figure 38. H&E staining of the excised organs and tumors of the mice-bearing Hs578t tumor. Representative histological images using H&E stain for visualizing the tumor, heart, kidney, liver and lung tissues from each group. Scale bar = 100 µM. 87 Part II - Therapeutic efficacy The second part of the experiment was to evaluate the in vivo antitumor efficacy of the DOX loaded mSLN (EBR85), which seems to display the best features. The abovementioned tumor implantation procedure was followed. Mice-bearing Hs578t tumors were randomly divided into 6 groups, which received specific treatments via i.v. injection: Group 1 received a saline solution, Group 2 was treated with EBR85 (1 mgDOX.Kg-1), Group 3 received the EBR89 nanoparticle (0.012 mgFe.Kg-1), the reference compound DOX (1 mgDOX.Kg-1) was administered to Group 4, and Groups 5 and 6 received EBR85 (1 mgDOX.Kg-1) and EBR89 (0.012 mgFe.Kg-1), respectively, followed by 1 h of hyperthermia. DOX belongs to class III of chemotherapeutic drugs being characterized for its high solubility and low permeability. So, the intravascular injection is the preferential route of administration to avoid cardiovascular pathology. Due to the compromised immune system of NSG mice, the DOX dose was chosen carefully and based on different articles. As demonstrated in Wunderlich, M., et al ., Mohanty, S., et al , Si, Y., et al ., and Favreau-Lessard, A., et al ., 3 mgDOX.Kg1 mice of accumulative dose seems to be the best option to observe a therapeutic effect without affecting the animals wellbeing (78,216–218). Tumor progression was evaluated once a week after finishing the treatment (Figure 12, Chapter 2, section 6). The animal survival rate was 100% with all animals reaching the predetermined 39-day endpoint. 88 Figure 39. Evaluation of nanoparticles’ therapeutic effect on mice-bearing Hs578t tumors. The 6 groups of mice were treated with vehicle, free DOX, EBR85, EBR89, or 1h of hyperthermia plus EBR85 or EBR89, three times once per week and humanely euthanized 39 days post-tumor implantation. ( A) Graphical representation of tumor evolution until two weeks after the last day of treatment, and (B) effect of the treatment on tumor growth along time, until the last day of experiment. (C) Graphical representation of tumor daily growth, on (D) is represented the percentage of vascularization on tumor region for each animal-treated. On (E) is shown the weight of tumors collected 39 days post-implantation, and on (F) is the graphical representation of the body weight of the animals throughout the experiment. * p <0.05, ** p <0.01, *** p <0.001 89 Tumor volume was evaluated for 22 days post-treatment through VEVO3100 ultrasound. At the end of the experiment, no significant differences were observed between treated groups, as observed Figure 39 (B) and Figure 40. The tumor volume increased along time in all experimental groups. Once again, hyperthermia treatment did not inhibit the tumor growth (as observed on EBR89+HT group), a result previously indicated in the first in vivo task. The combination of thermochemotherapy resulted in a lower tumor volume along the experiment (although not statistically significant at day 39 endpoint), which indicates a possible recovery of the tumor growth some days after treatment. When looking at tumor volume only two weeks after the last day of treatment (Figure 39 (A)), a significant difference on tumor volume of DOX-loaded EBR85+HT group is observed in comparison to unloaded EBR89+HT groups ( p <0.001), vehicle ( p <0.05), and EBR85 ( p <0.05). The dual-treatment, until 11-days post-treatment, showed a significant inhibitory effect on tumor growth, suggesting mSLNs efficacy to convert the electromagnetic energy from MH into heat energy, which enhances the therapeutic effect. Additionally, MH application improved the susceptibility of cancer cells to chemotherapy (already stated in (195)), as observed among DOX-loaded EBR85-treatment and thermochemotherapy EBR85-treatment ( p <0.05). 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