Biological effects of polyacrylic acid-coated and non-coated superparamagnetic iron oxide nanoparticles in in vitro and in vivo experimental models

Biological effects of polyacrylic acid-coated and non-coated superparamagnetic iron oxide nanoparticles in in vitro and in vivo experimental models Diana Manuel Mocho de Bastos Couto Tese do 3ºCiclo de Estudos conducente ao grau de Doutor em Ciências Farmacêuticas na especialidade de Química Farmacêutica e Medicinal apresentada à Faculdade de Farmácia da Universidade do Porto Thesis of the 3rd Cycle of Studies for obtaining the PhD degree in Pharmaceutical Sciences in the specialty of Pharmaceutical and Medicinal Chemistry submitted to the Faculty of Pharmacy of the University of Porto Work performed under the supervision of: Professora Doutora Eduarda das Graças Rodrigues Fernandes Professor Doutor Félix Dias Carvalho April 2015 É autorizada a reprodução integral desta tese apenas para efeitos de investigação, mediante declaração escrita do interessado, que a tal se compromete. Assinatura do autor, ii “Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world” Louis Pasteur iii To my parents To my sister To my nephew Alexandre iv ACKNOWLEDGEMENTS During this period of my life, many were the people that supported me and contributed to my achievements. Although it is impossible to mention and thank to all of these people, I could not leave without thanking to some of them, which revealed to be a true inspiration, a precious help and a source of encouragement to me: Ao Professor Doutor José Luís Costa Lima, não só por me ter permitido a integração no seu grupo de trabalho, ainda no antigo Departamento de Química Física e, mais recentemente Química Aplicada, como também por ter sempre colocado à minha disposição todos os meios, equipamentos e reagentes que necessitei para a realização deste trabalho. Não queria deixar de agradecer a simpatia com a qual sempre me tratou, os conselhos turísticos que me deu, assim como destacar o seu grande empenho e dedicação no que toca à gestão e ao bom funcionamento do departamento. Por fim, queria agradecer o facto de permitir que todas as deslocações e participações em congressos fossem possíveis. À Professora Doutora Eduarda Fernandes, por me ter recebido como sua aluna, numa fase inicial de iniciação à investigação e, posteriormente, como aluna de doutoramento. Dona de um grande rigor científico e académico e de um precurso profissional de valor indiscutível, agradeço a transmissão de conhecimentos, assim como a disponibilidade, a prontidão na discussão de resultados e revisão do trabalho e as oportunidades que me concedeu. Agradeço também, como não poderia deixar de ser, a orientação e o facto de sempre ter colocado no meu caminho as pessoas certas no momento certo. Ao Professor Doutor Félix Carvalho, meu co-orientador, por ter aceitado coorientar o meu trabalho, enriquecendo-o sempre com as suas ideias preciosas e a sua supervisão rigorosa, fruto de uma experiência profissional vasta e repleta de êxitos. Agradeço a transmissão de conhecimentos, a disponibilidade, a prontidão e o incentivo transmitido ao longo de todo este precurso. v À Marisa Freitas, com quem partilhei o laboratório durante estes quatro anos, um muito obrigado por toda a ajuda dispensada a todos os níveis: pelo acolhimento, pela amizade, pelos conselhos dados, por estar sempre perto quando necessário, pela discussão sempre atempada dos resultados, pela revisão do trabalho, pelas suas opiniões sempre válidas e preciosas, por ter feito tudo o que estava ao alcance dela e ainda mais… Por ter sido, sem dúvida, uma peça chave neste doutoramento… À Daniela Ribeiro, minha colega de curso e de doutoramento, agradecer todo o companheirismo, disponibilidade, acessibilidade e ajuda sempre prestada ao longo de todo este tempo. Ao Renan Chisté, pela ajuda prestada, amizade e alegria transmitida ao longo destes 4 anos de doutoramento. Aos restantes, atuais e antigos, colegas da FRAU (Free Radicals and Antioxidant Unit), um muito obrigada pela amizade, companheirismo e simpatia. À Engenheira Manuela Barros e Patrícia Monteiro pela simpatia e prontidão em satisfazer as nossas necessidades. Ao Professor Doutor Agostinho Almeida, assim como à Anne-Sophie Alves, pela valiosa ajuda e contribuição nos ensaios do doseamento de ferro. À Vera Costa, do Laboratório de Toxicologia, por me ter introduzido ao mundo da experimentação animal, e por o ter feito de uma forma exímia. Um muito obrigado por toda a ajuda prestada em todo o planeamento e execução dos estudos in vivo integrantes desta tese, assim como pela revisão do respectivo trabalho. vi Aos restantes membros dos Departamentos de Química Aplicada e Toxicologia, agradeço a simpatia, disponibilidade e ajuda sempre prestada. A los Profesores Doctores M. Arturo Lopez-Quintela, José Rivas y Paulo Freitas, del International Iberian Nanotechnology Laboratory por su contribución en la síntesis y caracterización físicoquímica de las nanopartículas de óxido de hierro, objeto de estudio de esta tesis. Muchas gracias a Carlos Gonzalez, así como a los otros colegas del International Iberian Nanotechnology Laboratory, por toda la ayuda prestada en los experimentos de caracterización de las nanopartículas. À Professora Doutora Graça Porto, do Hospital Geral de Santo António, pela sua contribuição para a realização deste trabalho e por ter permitido a obtenção das amostras de sangue, indispensáveis à realização deste. Às enfermeiras do Hospital Geral de Santo António pela prontidão com que sempre me disponibilizaram as amostras de sangue necessárias à realização deste estudo, assim como que aos dadores de sangue deste mesmo hospital, que generosamente contribuíram para este trabalho com a sua dádiva de sangue. À Professora Doutora Beatriz Porto, do Instituto de Ciências Biomédicas Abel Salazar, assim como às suas colaboradoras Rosa Sousa e Lara Andrade, por me terem recebido tão bem no seu laboratório de citogenética e me despertarem o interesse pela citogenética, uma área para mim desconhecida até então. Obrigada por toda a ajuda prestada, assim como pela amizade, acessibilidade e pelos bons momentos passados… vii À Professora Doutora Paula Silva, do Instituto de Ciências Biomédicas Abel Salazar, pela simpatia, acessibilidade, assim como por toda a ajuda prestada nos ensaios de histologia realizados no âmbito desta tese. À Doutora Margarida Lima, assim como à Magdalena Leander, do Hospital Geral de Santo António, pela ajuda prestada nos ensaios de citometria de fluxo e por terem colocado à minha disposição não só as instalações e equipamentos do Serviço de Hematologia Clínica, assim como também o seu conhecimento. Ao Professor Doutor José Alberto Duarte, da Faculdade de Desporto da Universidade do Porto, por toda a simpatia, ajuda, prestabilidade e por ter colocado à minha disposição as instalações do seu laboratório para a realização dos ensaios de histologia que são parte integrante desta tese. À D. Celeste Resende, da Faculdade de Desposto da Universidade do Porto, por toda a prestabilidade, amizade e pela ajuda valiosa nos ensaios de histologia realizados no âmbito desta tese. Ao Bruno Silveira pelo apoio gráfico na elaboração da tese. À Andreia Ferreira, Helena Gonçalves, Sanjay Radia, Vanessa Lima e Paulo Santos, pioneiros no que toca à imigração, por toda a amizade, partilha das vossas experiências de trabalho e/ou doutoramento, encontros e reencontros, em Portugal e no estrangeiro, e incentivo constante… Por me acolherem sempre em vossas casas e por estarem sempre presentes na minha vida mesmo quando estão ausentes. À Ana Isabel Couto, Daniela Sampaio, Florbela Santos, Joana Magalhães, Joana Sampaio, João Costa, Raquel Magalhães e Raquel Rodrigues, meus grandes amigos e viii INDEX ACKNOWLEDGEMENTS ……………………………………………………………………… v ABSTRACT ……………………………………………………………………………………… xi RESUMO ………………………………………………………………………………………...xiii INDEX …………………………………………………………………………………………… xv INDEX OF FIGURES ……………………………………………………………………….... xvii INDEX OF TABLES ……………………………………………………………………….… xxiii LIST OF ABBREVIATIONS AND CHEMICAL SYMBOLS ……………………………... xxv OUTLINE OF THE DISSERTATION ………………………………………………….…… xxix CHAPTER I. GENERAL INTRODUCTION I.1. Theoretical background …………………………………………………………………….. 1 I.1.1. Iron oxide nanoparticles: an insight into their biomedical applications ……………… 2 I.1.2. Toxicological and pro-inflammatory mechanisms of iron oxide nanoparticles ……. 64 I.2. General and specific objectives of the dissertation …………………………………… 130 CHAPTER II. ORIGINAL RESEARCH II.1. Interaction of polyacrylic acid coated and non-coated iron oxide nanoparticles with human neutrophils…………………………………………………………………..………… 133 II.2. Polyacrylic acid-coated and non-coated iron oxide nanoparticles induce cytokine activation in human blood cells through TAK1, p38 MAPK and JNK pro-inflammatory pathways ………………………………………………………………………………………. 146 xv II.3. Polyacrylic acid coated and non-coated iron oxide nanoparticles are not genotoxic to human T lymphocytes ………………………………………………………………………... 158 II.4. The biodistribution of polyacrylic acid-coated iron oxide nanoparticles implies a proinflammatory effect and some degree of liver toxicity …………………………………..… 166 CHAPTER III. INTEGRATED DISCUSSION AND CONCLUSIONS III.1. Integrated discussion …………………………………………………………………… 202 III.2. Conclusions ………………………………………………………………………….….. 210 III.3. References …………………………………………………………………………..….. 212 xvi INDEX OF FIGURES CHAPTER I. GENERAL INTRODUCTION I.1.1. Figure 1: Most widely coatings used in IONs. ……………………………………………… 10 I.1.2. Figure 1: Possible mechanisms underlying the cytotoxic effects of IONs (ATP: Adenosine triphosphate; Cyt c: Cytochrome c; IONs: Iron oxide nanoparticles; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor α). ..... 65 Figure 2: IONs-induced production of ROS and RNS (H2O2: Hydrogen peroxide; HO•: Hydroxyl radical; HOCl: Hypochlorous acid; iNOS: Inducible nitric oxide synthase; IONs: Iron oxide nanoparticles; NADPH: Nicotinamide adenine dinucleotide phosphate; •NO: Nitric oxide radical; NOS: Nitric oxide synthase; 1O2: Singlet oxygen; O2•-: Superoxide radical; ONOO-: Peroxynitrite anion). ………………………………………………….…..… 67 Figure 3: IONs influence on intrinsic and extrinsic apoptotic pathways (AIF: Apoptosis inducing factor; Apaf 1: Apoptotic protease-activating factor 1; FADD: Fas-associated death domain; IONs: Iron oxide nanoparticles; PS: Phosphatidylserine; TNF-α: Tumor necrosis factor α; TNFR: Tumor necrosis factor receptor; TRADD: Tumor necrosis factor receptor associated domain; Xkr8: Xk-receptor protein 8). ………………………..………. 94 xvii CHAPTER II. ORIGINAL RESEARCH II.1. Figure 1: Neutrophils’ oxidative burst in cells exposed to (A) PAA-coated ION (4–100 μg/mL) and (B) non-coated ION (4–100 μg/mL) in the absence and presence of DPI, at 37°C for 24 h. *p < 0.05 and ****p < 0.0001 comparatively to control (without ION), ɩɩp < 0.01 comparatively to 100 μg/mL PAA-coated ION and ɩɩɩp < 0.001 and ɩɩɩɩp < 0.0001 comparatively to 20 μg/mL non-coated ION. Data are expressed as percentage of neutrophil activation. Values are given as mean ± SEM (n ≥ 4). ………………………... 137 Figure 2: Neutrophils’ apoptosis assessed by microscopic morphology after exposure to PAA-coated and non-coated ION (4–100 μg/mL) at 37°C: (A) 16 h and (B) 24 h. *p < 0.05, **p < 0.01 and ****p < 0.0001 comparatively to control (without ION). Data are expressed as percentage of apoptosis relatively to control. Values are given as mean ± SEM (n ≥ 4). ……………………………………………….………………………………………………….. 137 Figure 3: Neutrophils’ apoptosis assessed by microscopic morphology at 16 h: without ION (A), with 100 μg/mL PAA-coated ION (B) and 100 μg/mL non-coated ION (C) (amplification 40×). Arrowheads indicate apoptotic neutrophils. ………………………… 138 Figure 4: Neutrophils’ apoptosis assessed by flow cytometry after exposure to (A) PAAcoated ION (4–100 μg/mL) and (B) non-coated ION (4–100 μg/mL) at 37°C for 16 h. *p < 0.05, **p < 0.01 and ***p < 0.001 comparatively to control. Data are expressed as percentage of annexin-V(+)/PI(−) cells. Values are given as mean ± SEM (n ≥ 4). …... 138 Figure 5: Flow cytometric analysis of annexin V binding assay. Neutrophils incubated at 37°C for 16 h without ION (A—black area) and with 100 μg/mL PAA-coated (B—green curve) and non-coated (C—blue curve) ION. ……………………………………………… 139 Figure 6: p53 activation in neutrophils exposed to PAA-coated ION (4–100 μg/mL) at 37°C for 24 h. *p < 0.05 comparatively to control (without ION). Data are expressed as percentage of p53 activation relatively to control. Values are given as mean ± SEM (n ≥ 4). …………………………………………………………………………………………….… 139 xviii Figure 7: Caspase 3 activity in neutrophils exposed to PAA-coated and non-coated ION (4–100 μg/mL) at 37°C: (A) 16 h and (B) 24 h. *p < 0.05, **p < 0.01, ***p < 0.001 comparatively to control (without ION). Data are expressed as percentage of caspase 3 activation relatively to control. Values are given as mean ± SEM (n ≥ 4). ……………… 139 Figure 8: Caspase 8 activity in neutrophils exposed to PAA-coated ION (4–100 μg/mL) at 37°C for 16 and 24 h. *p < 0.05 and **p < 0.01 comparatively to control. Data are expressed as percentage of caspase 8 activation relatively to control (without ION). Values are given as mean ± SEM (n ≥ 4). ……………………………………………….… 140 Figure 9: Caspase 9 activity in neutrophils exposed to PAA-coated and non-coated ION (4–100 μg/mL) at 37°C: (A) 16 h and (B) 24 h. **p < 0.01 and ***p < 0.001 comparatively to control (without ION). Data are expressed as percentage of caspase 9 activation relatively to control. Values are given as mean ± SEM (n ≥ 4). ……………………….… 140 Figure 1 Supplementary: TEM image and size distribution for non-coated ION particles dispersion with a mean particle size (± standard deviation) of 9.9±2.3 nm. ……………. 143 Figure 2 Supplementary: TEM image and size distribution for PAA-coated ION particles dispersion with a mean particle size (± standard deviation) of 10.1±2.4 nm. ………..… 143 Figure 3 Supplementary: pH dependence of the surface charge for the non-coated and PAA-coated ION particles dispersed in water. …………………………………..………… 144 Figure 4 Supplementary: [NaCl] dependence of the surface charge for the non-coated and PAA-coated ION particles dispersed in water. ……………………………………….. 144 Figure 5 Supplementary: ION dispersed in RPMI 1640 medium: (A) PAA-coated ION and (B) non-coated ION. …………………………………………………………..………… 145 II.2. Figure 1: IL-1β activation following exposure to: a PAA-coated ION (0.4–4 μg/mL), b noncoated ION (0.4–4 μg/mL), c PAA-coated ION (4 μg/mL) in the presence of inhibitors and d non-coated ION (4 μg/mL) in the presence of inhibitors, at 37 °C, for 24 h. ****p < 0.0001 and ***p < 0.001 when compared to control (without ION) and ηηηηp < 0.0001, ηηηp xix < 0.001 ηηp < 0.01 and ηp < 0.05 when compared to control (ION 4 μg/mL). Values are given as mean ± SEM (n ≥ 4) ……………………………………………………………….. 150 Figure 2: TNF-α activation following exposure to: a PAA-coated ION (0.4–4 μg/mL), b non-coated ION (0.4–4 μg/mL), c PAA-coated ION (4 μg/mL) in the presence of inhibitors and d non-coated ION (4 μg/mL) in the presence of inhibitors, at 37 °C, for 24 h. *p < 0.05 when compared to control (without ION) and ηηp < 0.01 and ηp < 0.05 when compared to control (ION 4 μg/mL). Values are given as mean ± SEM (n ≥ 4) ………………………. 150 Figure 3: IL-6 activation following exposure to: a PAA-coated ION (0.4–4 μg/mL), b noncoated ION (0.4–4 μg/mL), c PAA-coated ION (4 μg/mL) in the presence of inhibitors and d non-coated ION (4 μg/mL) in the presence of inhibitors, at 37 °C, for 24 h. ***p < 0.001 and **p < 0.01 when compared to control (without ION) and ηηηηp < 0.0001, ηηηp < 0.001, ηηp < 0.01 and ηp < 0.05 when compared to control (ION 4 μg/mL). Values are given as mean ± SEM (n ≥ 4) ………………………………………………………………………….. 151 Figure 4: IL-8 activation following exposure to: a PAA-coated ION (0.4–4 μg/mL), b noncoated ION (0.4–4 μg/mL), c PAA-coated ION (4 μg/mL) in the presence of inhibitors and d non-coated ION (4 μg/mL) in the presence of inhibitors, at 37 °C, for 24 h. ****p < 0.0001, ***p < 0.001 and **p < 0.01 when compared to control (without ION) and ηηηηp < 0.0001, ηηηp < 0.001 and ηηp < 0.01 when compared to control (ION 4 μg/mL). Values are given as mean ± SEM (n ≥ 4) ……………………………………………………………….. 151 Figure 5: IFN-γ activation following exposure to: a PAA-coated ION (0.4–4 μg/mL), b noncoated ION (0.4–4 μg/mL), c PAA-coated ION (4 μg/mL) in the presence of inhibitors and d non-coated ION (4 μg/mL) in the presence of inhibitors, at 37 °C, for 24 h. ***p < 0.001 and **p < 0.01 when compared to control (without ION) and ηηηηp < 0.0001, ηηp < 0.01 and ηp < 0.05 when compared to control (ION 4 μg/mL). Values are given as mean ± SEM (n ≥ 4) ……………………………………………………………………………………………….. 152 Figure 6: IL-10 activation following exposure to: a PAA-coated ION (0.4–4 μg/mL), b noncoated ION (0.4–4 μg/mL), c PAA-coated ION (4 μg/mL) in the presence of inhibitors and d non-coated ION (4 μg/mL) in the presence of inhibitors, at 37 °C, for 24 h. ****p < 0.0001 and **p < 0.01 when compared to control (without ION). Values are given as mean ± SEM (n ≥ 4) ……………………………………………………………………..………….…152 Figure 7: Inflammatory pathways triggered by the studied PAA-coated and non-coated ION ………………………………………………………………………………………...…… 154 xx II.3. Figure 1: Cell cycle distribution (expressed in percentage) following exposure to (A) PAAcoated and (B) non-coated ION (4, 20 and 100 µg/mL), at 37ºC, for 48 h. Values are given as mean of the percentages of T lymphocytes in the phases G0/G1, S and G2/M (n ≥ 7). ……………………………………………………………………………….………………..… 161 Figure 2: Typical chromosomal aberrations observed in the present study (amplification 100x): metaphase without any chromosomal aberration (A); metaphase with a gap (B), indicated by a black arrowhead; metaphase with dicentric chromosomes and acentric fragments (C), indicated by black and blue arrowheads, respectively; metaphase with a figure, breaks and an acentric fragment (D), indicated by black, orange and blue arrowheads, respectively; metaphase with rings and a break (E), indicated by black and orange arrowheads, respectively; pulverized metaphase (F). …………………………... 162 Figure 3: Aberrant cells (expressed in percentage) in the presence of BLM (10 μg/mL) and: PAA-coated ION (4, 20 and 100 µg/mL) and non-coated ION (4, 20 and 100 µg/mL), at 37ºC, for 48 h. Values are given as mean ± SEM (n = 12). …………………………… 163 Figure 4: Mean number of breaks per cell following exposure to BLM (10 μg/mL) and: PAA-coated ION (4, 20 and 100 µg/mL) and non-coated ION (4, 20 and 100 µg/mL), at 37ºC, for 48 h. Values are given as mean ± SEM (n = 12). ……………………………… 163 II.4. Figure 1: Iron biodistribution (liver, spleen, tail, lungs and heart) in CD-1 mice administered with PAA-coated IONs (0, 8, 20 or 50 mg/kg, i.v.) 24 hours before. ***p<0.001, **p<0.01 and *p<0.05 when compared to control (mice administered with 0.9% saline solution). Data are expressed as organ iron (μg iron/g of organ). Values are given as mean ± SEM (n = 6). …………………………………………………………….… 191 Figure 2: Light micrographs of paraffin sections from mouse liver stained with haematoxylin-eosin (A, C and E) and Perl’s Prussian blue (B, D and F). No iron was detected in control animals with both stainings (E and F). In animals exposed to PAAxxi coated IONs (A, B, C and D), iron-loaded Kupffer cells (thin arrows) in sinusoids (thick arrows - endotheliocytes) were observed. Under lower magnification, it is possible to detect the abundance of the iron in the liver of treated animals both with haematoxylineosin (A) and with Perl’s Prussian blue (B). Boxed area in A is shown at higher magnification on image below (C), where it is possible to observe that, with this staining, the Kupffer cell cytoplasm has a granular and golden brown appearance. Binucleate hepatocytes (bH) are common in the mice. ………………………………………………... 192 Figure 3: Light micrographs of paraffin sections from liver of mice exposed to PAA-coated IONs stained with haematoxylin-eosin. Under lower magnification (A) clusters of the early necrotic hepatocytes (eNH) were identified by an increase of eosinophilia. Boxed area is shown at higher magnification on the right (B). At this magnification, it is possible to observe a local distribution pattern of eosinophilic hepatocytes and also the presence of iron-loaded Kupffer cells (thin arrows). ……………………………………………..……… 193 Figure 4: Mice spleen. With Masson's trichrome staining (A, B and C), both control and PAA-coated IONs mice spleens showed a normal morphology (A), with a smaller proportion of red pulp (R). In mice, the splenic red pulp (B) is a major site of myeloid, erythroid hyperplasia and megakaryocytic hyperplasia (Meg - megakaryocyte). As shown in C, the white pulp (W) is a lymphoid area consisting of sheaths of lymphoid cells composed primarily of T cells (T) around the central arteriole (A). No iron was detected in white pulp with the Perls’ blue staining, both in control and treated animals (50 mg/kg) (D). When compared with the control (E), the splenic red pulp had a great amount of ironladen macrophages in treated animals (50 mg/kg) (F). …………………………………... 194 Figure 5: Kidney of mice exposed to PAA-coated IONs stained with Masson’s trichrome. No histopathologic alterations were found in mice kidney after the treatment. Abbreviations are: PT – Proximal tubule; DT – Distal tubule; AT – Afferent arteriole; RSC - Renin-secreting cell; VP – Vascular polar; C – Renal corpuscle; BS – Bowman’s space; BC - Bowman’s capsule; P - Podocytes. …………………………………………………… 195 Figure 6: MDA evaluation in liver and kidney of CD-1 mice administered with PAA-coated IONs (0, 8, 20 or 50 mg/kg) 24 hours before. ***p<0.001 when compared to control (mice administered with 0.9% saline solution). Data are expressed as pmol MDA/mg protein. Values are given as mean ± SEM (n ≥ 5). …………………………………………….…… 196 xxii INDEX OF TABLES CHAPTER I. GENERAL INTRODUCTION I.1.1. Table 1: IONs available in the market. …………………………………………………..…. 28 Table 2: Recommended doses of IONs used for medical purposes. ………………….…. 29 I.1.2. Table 1: Toxic effects of IONs in several cell types and tissues. ………………………… 77 CHAPTER II. ORIGINAL RESEARCH II.3. Table 1: BLM (10 μg/mL) and PAA-coated ION-induced chromosome instability in lymphocyte cultures from 12 individuals. ……………………………………………….…. 164 Table 2: BLM (10 μg/mL) and non-coated ION-induced chromosome instability in lymphocyte cultures from 12 individuals. …………………………………………………... 164 xxiii II.4. Table 1: Plasma levels of AST, ALT and total CK in CD-1 mice exposed to PAA-coated IONs (0, 8, 20 or 50 mg/kg). ……………………………………………………..………….. 197 Table 2: Leukocyte differential counts for mice injected with PAA-coated IONs (0, 8, 20 or 50 mg/kg). ...................................................................................................................... 198 Table 3: Body and organs weight of CD-1 mice administered with PAA-coated IONs (0, 8, 20 or 50 mg/kg). ………………………………………………………………………………. 199 Table 4: GSHt, GSSG, GSH, ATP levels and GSH/GSSG ratio of CD-1 mice exposed to PAA-coated IONs (0, 8, 20 or 50 mg/kg). ………………………………………………….. 200 xxiv CHAPTER I GENERAL INTRODUCTION I.1. Theoretical background 1 I. General Introduction I.1.1. IRON OXIDE NANOPARTICLES: AN INSIGHT INTO THEIR BIOMEDICAL APPLICATIONS Manuscript accepted for publication in Current Medicinal Chemistry 2 Iron oxide nanoparticles: an insight into their biomedical applications Diana Coutoa, Marisa Freitasa, Félix Carvalhob*, Eduarda Fernandesa* aUCIBIO-REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal bUCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal Corresponding authors: Eduarda Fernandes, PharmD; PhD UCIBIO-REQUIMTE, Laboratory of Applied Chemistry Department of Chemical Sciences Faculty of Pharmacy, University of Porto, Porto, Portugal Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal Phone: +351 220428675 Email: [email protected] Félix Carvalho, PharmD; PhD UCIBIO-REQUIMTE, Laboratory of Toxicology Department of Biological Sciences Faculty of Pharmacy, University of Porto, Porto, Portugal Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal Phone: +351 220428600 Email: [email protected] I. General Introduction 3 Abstract Iron oxide nanoparticles (IONs) are among the most common types of nanoparticles (NPs) used in biomedical applications. IONs can be presented in different forms [e.g. magnetite (Fe3O4), hematite (α-Fe2O3) and maghemite (γFe2O3)], and are usually coated with substances and/or polymers according to the purpose for which they are intended to be used. In recent years, IONs use has been increasing exponentially in many fields of biomedicine, namely in magnetic resonance imaging, cell sorting, tissue repair, induction of hyperthermia and drug delivery, among others. This review aims to provide an update on the different IONs and the substances and/or polymers that can be used to coat the IONs core as well as their applications and biological properties, namely their biodistribution in the human body and their cellular internalization pathways. Keywords: Biodistribution; biomedical applications; coatings; internalization routes; iron oxide nanoparticles; toxicity. I. General Introduction 4 Contents 1. Introduction 2. Types of IONs 3. Sizes of IONs 4. Coatings 4.1. Types of coatings 4.1.1. Polyvinyl alcohol (PVA) 4.1.2. Poly(vinylpyrrolidone) (PVP) 4.1.3. Polyethylene glycol (PEG) 4.1.4. Polyacrylic acid (PAA) 4.1.5. Dextran 4.1.6. Mannan 4.1.7. Poly(γ-glutamic acid) (PGA) 4.1.8. Chitosan 4.1.9. Dimercaptosuccinic acid (DMSA) 4.1.10. Inorganic NPs 4.1.10.1. Silica and aminosilane 4.1.11. Polycationic transfection agents (PTA) 4.1.12. Folic acid 4.2. Toxicity of coatings 5. Magnetization of IONs 6. Biodistribution 6.1. Route 6.2. Influence of size 6.3. Half-life 6.4. Circulation and homeostasis 6.5. Elimination 6.6. Differences between normal and cancer cells 6.7. Cellular localization 7. Internalization routes 7.1. Transcytosis 7.2. Endocytosis 7.2.1. Phagocytosis 7.2.2. Macropinocytosis 7.2.3. Clathrin-mediated endocytosis I. General Introduction 5 7.2.4. Caveolae-mediated endocytosis 7.2.5. Receptor-mediated endocytosis 7.2.5.1. Transferrin receptor 7.2.5.2. Scavenger receptor (SR)-A 7.2.5.3. Mac-1 receptor 7.2.5.4. Other receptors 8. NPs available in the market 9. Concentration 10. Applications 10.1. Magnetic resonance imaging (MRI) 10.1.1. Labeling and imaging 10.1.2. Cell sorting 10.2. Tissue repair 10.3. Drug delivery 10.4. Hyperthermia 10.5. Transfection 10.6. DNA detection 10.7. Tissue soldering 10.8. Biofilm treatment and antibacterial activity 10.9. Vaccine carriers 10.10. Peroxidase activity 11. Final notes 12. List of Abbreviations 13. Conflicts on Interest 14. Acknowledgements 15. References I. General Introduction 6 1. Introduction Nanotechnology encompasses structures of 100 nm or smaller that may present several dimensions: nanoscale in one dimension (very thin surface coating), in two dimensions (nanotubes and nanowires) or in three dimensions, known as nanoparticles (NPs), which comprise nanomaterials. Nanomaterials are present in toothpastes, sunscreens, food products and even sanitaryware coatings. Currently, the use of nanomaterials is being extended to multiple areas such as cell labeling, gene delivery, drug targeting, biosensors, chemistry, material science, physics, medicine, microelectronics and hyperthermia therapy [1, 2, 3, 4] NPs can present different characteristics and be engineered from almost all chemical substances. They have physical and chemical properties that differ from the atom or bulk counterparts and have been recognized to have great potential in several fields due to the unique properties conferred by their surface-area-to-volume ratios [5, 6, 7], namely: -the ease with which they are captured by the cells; -avoidance of clearance by the immune system compared to traditional macroscale materials; -porous structure; -lower melting points; -increased light absorption. Iron oxide nanoparticles (IONs) are among the most common types of NPs used in medical applications. IONs can exist in different forms and are usually coated with substances and/or polymers that are able to confer the expected properties according to the purpose for which they are intended. Their small dimensions make IONs more chemically reactive with particle size being inversely proportional to bioactivity and toxicity [8, 9, 10]. From a health and environmental perspective, this can be harmful if the NPs are toxic or in some way disturb biological functions and lead to undesirable health effects. In particular, people with pre-existing medical conditions are at greater risk [11, 12]. This review aims to provide an update on the different types of IONs and the substances and/or polymers that can be used to coat the IONs core as well as their applications and biological properties, namely their biodistribution in the human body and their internalization pathways into cells. I. General Introduction 7 2. Types of IONs IONs are particles with dimensions ranging from 1 to 100 nm, composed of iron oxide. IONs consist of a magnetic iron oxide core with a coating of non-magnetic surface chemistry and exist in many forms in nature, namely magnetite (Fe3O4), hematite (αFe2O3) and maghemite (γ-Fe2O3) [7, 9]. Magnetite is a mixture of FeO and Fe2O3 containing both Fe3+ and Fe2+ ions, with a cubic inverse spinel structure. It is thermodynamically unstable due to the oxidation of magnetite (from Fe2+ into Fe3+) to form maghemite in the presence of moisture, air and light [13, 14, 15, 16, 17]: Fe3O4+2H+  γ-Fe2O3+Fe2++H2O (Equation 1) Maghemite NPs (γ-Fe2O3) are composed of fully oxidized cubic crystals and are therefore extremely stable under aerobic conditions even when bacteria are present [14, 18]. Hematite (α-Fe2O3) is a mineral form of Fe3+ oxide presenting a rhombohedral corundum crystal structure. It is considered the most abundant iron oxide polymorph and its iron content is approximately 70%. Due to its application as a pigment, it is one of the most common industrially used forms of iron oxide, apart from magnetite [18, 19, 20, 21]. 3. Sizes of IONs IONs can be classified according to their size which greatly influences their magnetic and biological properties. Ultra-small superparamagnetic iron oxide nanoparticles (USPIONs) are less than 50 nm in diameter and comprise 2-3 nm single crystal iron oxide cores enclosed in a coating that makes them biocompatible. Particles with dimensions larger than 50 nm are denominated superparamagnetic iron oxide nanoparticles (SPIONs) [16, 22, 23, 24]. 4. Coatings Biomedical applications demand that the IONs present uniform chemical and physical properties and for that reason, have high magnetization values with a size lower than 100 nm being essential. The nature of the coating determines the total size of the colloid as well as the biodistribution and biokinetics of SPIONs in vivo [25]. I. General Introduction 8 Since introduction of the first contrast agents in magnetic resonance imaging (MRI) in the decade of 1980 [26], most of the SPIONs and USPIONs agents have been fabricated with different types of coating material including citrate, dextran, albumin, starch, polyethylene glycol (PEG) and silicones to achieve their dispersion status and uptake selectivity by macrophage or endothelial cells [27, 28]. The carboxylate group is the most common fixation site on an iron oxide core. Other anchoring agents have been researched, such as phosphate, sulfonate and phosphonates, despite their strong affinity towards the core of iron oxide [29, 30]. The utilization of bifunctional phosphonic acidbased coupling agents with polar end groups (-OH,-COOH,-NH2) not only makes the NPs hydrophilic and stable regarding aggregation, but also conveys functionality on the surface to provide easy access to bioconjugates [31]. These coatings ideally should be nonantigenic and nonimmunogenic, have a high affinity for the iron oxide core and impede opsonization by plasma proteins [32]. The coating of SPIONs is indispensable because it: -decreases the aggregation tendency of the non-coated NPs (which occurs due to their hydrophobic surfaces with large surface area to volume ratio and their van-der walls and magnetic dipole–dipole attractive forces [7, 33, 34]), thus improving their colloidal stability and dispersibility [35]; -prevents the oxidation of their surface [35], given that non-coated IONs tend to be transformed from magnetite (Fe3O4) to maghemite (γ-Fe2O3) [13]; -provides a surface for conjugation of targeting ligands and drug molecules such as proteins, antibodies, therapeutic genes, targeting ligands, etc. [13, 35, 36]; -augments blood circulation time by evading clearance from the reticuloendothelial system (RES) [35]; -renders the NPs biocompatible and decreases nonspecific interactions, consequently reducing toxicity [35, 37]; -increases their efficiency of internalization by target cells [35, 37]. 4.1. Types of coatings Diverse groups of coating materials are used to modify magnetic NPs surface chemistry, namely: -synthetic polymers, such as poly(ethylene-co-vinyl acetate), poly(vinylpyrrolidone) (PVP), poly(lactic-co-glycolic acid) (PLGA), PEG and polyvinyl alcohol (PVA); -natural polymers, such as dextran, pullulan, gelatin and chitosan; I. General Introduction 9 -organic surfactants, such as dodecylamine, sodium carboxymethylcellulose and sodium oleate; -inorganic metals, such as gold; -inorganic oxides, such as carbon and silica; -bioactive molecules and structures, such as liposomes, lipids, ligands/receptors and peptides [9, 13, 28, 38]. In this review, we refer only to the most relevant coatings described in the literature (figure 1). Figure 1. Most widely coatings used in IONs. 4.1.1. Polyvinyl alcohol (PVA) PVA is a water soluble synthetic polymer that has exceptional emulsifying, filmforming and adhesive properties. Besides the fact that PVA enhances polymer-surface interactions, PVA also forms hydrogen bonds between the polymer chains which results in a hydrogel structure that embeds the NPs and is responsible for steric stabilization above and around the critical polymer concentration. This structure prevents aggregation and I. General Introduction 10 receptor-mediated endocytosis and evade nonspecific capture of NPs in normal tissues, producing their apoptotic effect solely in cancer cells [95, 99]. 4.2. Toxicity of coatings Although coatings are beneficial for the above-mentioned reasons, there are increasing reports concerning their toxicity. After NPs core coating, the acute toxicity experimented is ascribed to the physicochemical properties of the NPs surface, which is key for cellular uptake and particle-cell interactions. These properties comprise surface charge, hydrodynamic radius and toxicity inherent to the coating materials [9, 66]. It is known that positively charged SPIONs are usually more toxic due to the fact that positive charges tend to be firmly attached to the cell surface, compared to the anionic NPs. This occurs because the resting cell membrane potential is negative [100]. Following intravenous injection of positively charged SPIONs, nonspecific interactions and unspecific adsorptive endocytosis take place, which results in aggregates formed with plasma proteins and blood cells. Therefore, cationic SPIONs will be more concentrated into the cells than anionic SPIONs. Cell membrane integrity was demonstrated to be more severely damaged and intracellular vesicles contained a higher concentration of SPIONs in the cells exposed to positively charged SPIONs [100]. Hoskins et al [101] reported that PEI-coated magnetic IONs decreased the viability of breast cancer (MCF-7), neuroblastoma (SH-SY5Y) and macrophage-like (U937) cell lines respectively. It was also reported that PEI-coated NPs increased reactive oxygen species (ROS) production which resulted in cellular stress [101]. The explanation for these toxic effects is that, by inserting the cationic SPIONs in an acidifying lysosomal compartment, the unsaturated amino groups are able to trap protons that are provided by the proton pump for cationic SPIONs digestion. One Clion and one water molecule per proton are propagated in the lysosome vesicle. Owing to Coulombic interactions, more protons will be injected into the lysosome which will cause lysosomal swelling and consequent rupture, leading to NPs deposition in the cytoplasm and lysosomal content spillage [102, 103]. Regarding polyethylene oxide (PEO), it was reported that the shortest tails of PEO are more toxic when compared to the longest tails. The shortest 0.75 kD tail triggered chromatin condensation, nuclear blebbing and formation of apoptotic bodies [16]. In the case of PAA, it was reported that PAA-coated IONs markedly reduced cell viability in OCTY mouse cells. Only 16% of cell viability was observed at an iron concentration of 400 µg/mL after 72 hours of culture [104]. I. General Introduction 17 5. Magnetization of IONs Superparamagnetism is a fundamental criteria of magnetic NPs in nanomedicine. This phemomenon arises from competition between thermal fluctuations of magnetic moments of NPs and the ordering effect on such NPs due to an external magnetic field [103]. When an external magnetic field is not present, ferromagnetic materials are not magnetized, given that the magnetization of the ferromagnetic domains is randomly oriented. Contrary to paraand diamagnetic materials, elimination of the magnetic field will not lead to magnetism loss, but the NPs will still display a remnant magnetization given that the large Weiss domains (regions within a magnetic material which have a uniform magnetization) are still aligned along the original field. When the particles size is smaller than the diameter of the Weiss/ferromagnetic domains (about 30 nm) leading to NPs not showing any magnetic remanence (i.e. restoration of the induced magnetization to zero upon elimination of the external magnetic field), superparamagnetism occurs. Superparamagnetic IONs are the most widely applied NPs in the biomedical field. Each of the NPs may be considered a monomagnet and is regarded as a fully magnetized single Weiss monodomain, which is a direct consequence of the spinel structure of the NPs, permitting strong magnetic coupling and therefore a perfect alignment of the individual magnetic spins. The IONs saturation magnetization diminishes with particule size, which is unfavorable for the efficiency (relaxivity) of the IONs. This decline of saturation magnetization has been ascribed to surface effects, namely spin canting, which is a lack of full alignment of the spins at the surface in high magnetic fields [23, 28, 103, 105]. 6. Biodistribution Biodistribution of IONs and their efficient delivery to a specific tissue depends on their chemical and physical characteristics. NPs may be characterized according to their surface coating, size, surface charge, density, surface hydrophobicity and pH of their suspensions [106]. These properties are particularly relevant and strongly influence blood circulation time, bioavailability and metabolism of the NPs within the body [9, 46]. The coating of IONs plays a relevant role in their biodistribution. Coating materials that restrict or hamper water access to the iron oxide core display significantly lower degradation rates, as demonstrated by the enhanced half-life of these NPs in the liver [107, 108]. The surface charge of SPIONs also plays a key role in their blood half-life. Cationic NPs tend to adhere unspecifically to cells. Strong negative charges result in increased liver uptake [109]. I. General Introduction 18 Furthermore, the biodistribution of NPs is influenced by a myriad of factors including the route of administration and the physiological environment to which NPs are introduced [110]. NPs could straightforwardly pass through diverse tissue compartments and move rapidly and freely to target organs [111]. The characteristic biodistribution of NPs is 80–90% in liver, 5–8% in spleen and 1–2% in bone marrow [9]. 6.1. Route Magnetic NPs may be administered into the body via several routes. Although the preferred route for cancer therapy is intravenous injection, other routes are considered effective for drug delivery. For example, in the case of lymphatic targeting, interstitial injections (namely intradermal and subcutaneous) are chosen, since low doses of magnetic NPs reach high accumulation levels in the regional lymph nodes [36]. 6.2. Influence of size Particle size plays a fundamental role in the biodistribution of IONs. In general, larger particles circulate in blood for shorter time periods than smaller particles. It was described that larger particles greater than 200 nm in diameter are usually entrapped by the liver and spleen due to mechanical filtration. IONs over 50 nm are usually captured quickly by the RES in Kupffer cells of the liver and have a restricted uptake in lymph and bone tissues. They are eventually eliminated by the phagocytic cells which results in diminished blood circulation times [9, 112, 113]. Due to their greater half-life, USPIONs can pass through the capillary wall and present more extensive tissue distribution. For this reason, they are readily captured by phagocytic cells, namely the Kupffer cells of the liver, mononuclear T cells and circulating monocytes/macrophages, as well as reactive astrocytes, dendritic cells and microglia inside the brain, and can be found in phagocytic cells in the lymph nodes, bone marrow, liver and spleen. This does not occur with SPIONs, which are found almost exclusively in the liver and spleen [114, 115]. However, smaller NPs of diameters less than 10 nm are quickly eliminated via extravasation and renal clearance. Particles between 10 to 100 nm are consequently ideal for subcutaneous injection and display the most prolonged blood circulation times (in humans of 24 to 36 h and in rats approximately 5 h [116]), having been reported to accumulate in bone marrow, heart, kidney, intestine, spleen and stomach [13, 117]. The ability of these IONs to cross the blood-brain barrier (BBB) and move into the central nervous system (CNS) via the olfactory pathway was also described as well as the ability to penetrate stratum corneum and hair follicles, attaining viable skin epidermis [9, 18]. The NPs in this size interval are I. General Introduction 19 sufficiently small to evade RES and in addition, enter the very small capillaries within the body tissues. For this reason, they may provide the most effective distribution in several tissues [13]. The capture of SPIONs into the RES is associated with protein adsorption on the particle surface and ensuing opsonization. Consequently, decreasing the particle size will reduce NPs phagocytosis, which results in a significant increase in plasma half-life and larger biodistribution [118, 119]. 6.3. Half-life For all IONs, the blood half-life is dose-dependent. This feature is related to a gradual saturation of macrophage capture by the liver or other macrophage-rich organs, namely spleen and bone marrow [28]. The blood half-life of IONs is normally higher in humans than in animals. Although it was described that SPIONs are more appropriate for primary monocytes labeling, USPION are more frequently used for in vivo labeling due to their long blood pool half-life: the plasma half-life for USPIONs has been reported to range from 80 minutes to more than 24 hours, compared with only ~2 to 4 hours for standard SPIONs [28, 114, 120]. Ferumoxides interaction time with cells is reported to be approximately 6 minutes in vivo compared to 4 h in vitro [65]. The large variability in USPIONs circulation time is related to the composition of the surface coating used to stabilize the particle. In general, the extended circulation time for USPIONs derives from the particles not being immediately recognized by phagocytic cells of the RES [114]. Ferumoxtran-10 (15-30 nm) is reported to have a longer blood half-life (around 30 h in humans) without accumulating in specific tissues compared to ferumoxide AMI-25 (120180 nm) [28]. 6.4. Circulation and homeostasis The destiny of NPs following intravenous administration is defined by their polymer coatings. After injection of NPs into the bloodstream, they are usually opsonized and this step is critical in dictating their outcome. Usually, opsonization makes the NPs recognizable by the RES. The liver macrophages and, to a lesser extent, the macrophages in circulation and of the spleen, play a significant role in the elimination of opsonized NPs [121]. IONs do not extravasate into the interstitium in most tissues. However, some extravasation was observed across the discontinuous endothelium of microvessels in the liver, spleen, lymph nodes and bone marrow [122]. In addition, when NPs are transported to the tumor site through the bloodstream, some extravasation can occur across hyperpermeable microvessels [123]. After clearing by the RES, there is the I. General Introduction 20 possibility that magnetic NPs recirculate in the blood or inflammatory lesions, being transported by membrane vesicles which are released by apoptotic macrophages. Consequently, the vesicular traffic permits a dynamic intercellular redistribution of NPs [124]. Intracellular iron homeostasis involves a tightly regulated balance between the synthesis of transferrin receptors and the synthesis of intracellular ferritin molecules, responsible for cellular iron capture [125, 126]. Clear evidence has now emerged that once the USPIONs are ingested by macrophages, they are dissolved within the lysosomes by a succession of hydrolyzing enzymes with the low pH (~ 4.5) of the lysosome, where this low pH and heme oxygenase divides the iron oxide core into iron ions. These ions are subsequently incorporated into the hemoglobin pool and potentially into other iron requiring proteins such as apoferritin, ferritin and transferrin [36, 115, 127, 128]. When cytoplasmic iron content rises, ferritin synthesis is triggered, which results in the entrapment of toxic iron (Fe2+) into the ferritin molecules [129]. However, it was described that elevated levels of free iron ions from magnetic NPs may trigger a disequilibrium in body homeostasis and lead to aberrant cellular responses including oxidative stress, DNA damage, epigenetic events and inflammatory processes [36]. 6.5. Elimination The efficacy of NPs clearance exerted by RES depends on the chemical composition, size and surface chemistry of the magnetic NPs and may influence mechanisms of particle internalization as well as macrophage activation [9]. The most desirable excretion pathway for IONs is via the kidney (89%) given that this route implies minimal intracellular catabolism and reduces the probability of producing reactive oxygen species (ROS) and therefore, associated toxicity. Renal excretion is considered the safest route of excretion for IONs. However, the hydrodynamic size, shape, surface charge and surface coating of IONs play a key role in the regulation of their renal clearance [28, 35, 130]. The rest of the iron is excreted slowly (84 days) in feces [131]. The presence of γFe2O3 NPs in podocytes has been reported, suggesting that they can cross the glomerular basement membrane and consequently, are filtered into the urine. This is also supported by the finding that NPs are present in lysosomes/endocytic vesicles in the cytoplasm of proximal tubular cells [132]. Regarding coating elimination, it has been suggested that the acidic environment of the lysosomal compartment (pH ~ 5.5) may cause the rupture of the dextran shell I. General Introduction 21 (because of the existence of intracellular dextranases in macrophages) and liberate Fe3+ from IONs. Sulfatases are also lysosomal enzymes that break a variety of sulfated carbohydrates, being these compounds able to be used as IONs coating [133, 134]. 6.6. Differences between normal and cancer cells Carcinoma and normal cells were shown to have different metabolism regarding iron uptake. Breast carcinoma cell metabolism, for example, is faster than in normal cells. As a result, carcinoma cells necessitate larger amounts of micronutrients (namely iron), which can be evidenced by the increase of transferrin receptors in these cells [135, 136]. Moreover, in fast-growing tumors, neovascularization is habitually deficient and associated with poor lymphatic drainage, which permits increased permeation and retention of therapeutic nanodrugs [137]. However, it was described that SPIONs are not phagocytosed by reticuloendothelial cells in tumoral lymphoid tissues because macrophages are replaced by cancer cells and consequently, these tissues lack reticuloendothelial cells [138, 139]. 6.7. Cellular localization IONs have been described as having cytoplasmic localization in different cells, namely non-coated Fe3O4 NPs in HeLa cells [140], DMSA-coated Fe3O4 NPs in RAW264.7 cells [141, 142], pullulan-coated Fe3O4 NPs in fibroblasts [143] and citratecoated γ-Fe2O3 NPs in THP-1cells [124]. IONs seem to be grouped into clusters in the cytoplasm of the cells, in part around the nucleus and possibly localized in endocytotic vesicles (for example lysosomes) [124, 144, 145]. It was reported that in BEAS-2B cells, IONs are surrounded by mitochondria but do not have the capacity to penetrate them [145]. However, in monocytes NPs are described as entering into the mitochondria, having been found on the membrane and cristae, sometimes filling the matrix [146]. 7. Internalization routes Diverse cellular internalization routes exist which include transcytosis and endocytosis (clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis and macropinocytosis), among others. It is believed that all of these types of mechanisms are used by cells to internalize several substrates [9, 147]. The cellular internalization routes through which NPs enter into the cells depend on their surface properties which may be characterized according to their I. General Introduction 22 hydrophilic/hydrophobic features, chemistry or surface energy. These surface characteristics determine the means by which NPs adsorb to the cell surfaces and more specifically, dictate the behavior of the cells in contact with them [46]. Size is also determinant for NPs internalization and efficacy of cellular uptake. For NPs in the interval 10–30 nm, diffusion through membrane channels or across plasma membranes may occur [36]. Smaller NPs may also be internalized by pinocytosis while in the case of larger NPs, phagocytosis and receptor-mediated endocytosis seem to be the most common internalization pathways [148]. In phagocytic cells, smaller NPs are often internalized less effectively than larger NPs while in nonphagocytic T-cells, NPs with intermediate sizes were shown to be internalized more effectively than any other size [148]. 7.1. Transcytosis Transcytosis is described as being the vesicular transendothelial transport mechanism. Endothelial vesicles represent the most important mediator of macromolecular transport via the capillary wall and are increased in inflamed tissues [149, 150]. Endothelial transcytosis into the interstitium throughout the body has been described as a mechanism by which IONs uptake can occur. When USPIONs are in the bloodstream, they may cross the endothelium by transcytosis. Thereafter, USPIONs will be taken up by cells through endocytosis or other processes [28]. Transcapillary passage of the NPs through the venules into the medullary sinuses within the lymph nodes has been described, with subsequent phagocytosis by macrophages [151]. 7.2. Endocytosis Most inorganic NPs are captured by cells through the endocytic route. Internalization via endocytosis (the process of capturing macromolecules into cells by including them in membrane vesicles) after cell membrane contact can occur through a variety of mechanisms: clathrin-mediated endocytosis, caveolae-mediated endocytosis, receptor-mediated endocytosis, phagocytosis, pinocytosis and macropinocytosis [152]. In endocytosis, NPs interact with the cell plasma membrane which encloses and traps them into vesicles. These vesicles are denominated early endosomes and are transported via the cytoplasm and fuse sequentially with late endosomes and lysosomes. In this process of cell internalization, NPs meet compartments with various enzymatic activities and continuously decreasing pH, ending up in lysosomes [153, 154]. The endocytic pathway was previously reported for anionic magnetic NPs after interacting with the plasma membrane via electrostatic interactions [124, 155]. It was also described that I. General Introduction 23 PVA-coated SPIONs were endocytozed by monocyte-derived human dendritic cells in a dose-related manner, predominantly via an actin-dependent process [41]. Cellular uptake data showed that the uptake of long-circulating dextran-coated IONs was ever-present in several tumor cells and was not saturable, which suggests that fluid-phase endocytosis rather than receptor-mediated endocytosis is the uptake mechanism [156]. Improved endocytosis strategies may be sought, namely the use of transfection agents, antibody or receptor-mediated delivery or conjugated, cellular translocation signal peptides [4, 103]. 7.2.1. Phagocytosis Phagocytosis is an actin-dependent endocytic mechanism which involves phagocytes such as dendritic cells, macrophages and neutrophils [157]. It is often triggered by particle opsonization and ensuing F-actin-driven pseudopods receptormediated activation, which encloses the NPs in a phagosome present in the cytoplasm [158]. It was observed that phagocytic uptake of IONs increases when NPs size increases. USPIONs of sizes varying from 20–50 nm and monocrystalline IONs (MIONs) of 10–20 nm sizes were less efficiently phagocytized than SPIONs between 50–180 nm [115, 159]. Another study reported that phagocytosis was highest in response to 10 nm magnetite particles and minimal in response to 1 µm particles, which is not completely in line with the previous mentioned studies [160]. 7.2.2. Macropinocytosis Macropinocytosis is a highly conserved endocytic process where extracellular fluid and its contents are enclosed into cells through macropinosomes. These are heterogeneous and large vesicles formed through direct actin polymerization near the plasma membrane, causing membrane ruffling [157, 161]. Rejman et al [147] demonstrated that efficient uptake of microspheres requires cholesterol in their plasma membrane which can indicate that macropinocytosis is in some way involved in the internalization of larger sized particles. Macropinocytosis, although not ubiquitous, may function in cells other than macrophages. It was described that magnetic NPs can be incorporated into A549 human lung endothelial cells by macropinocytosis [12, 162]. 7.2.3. Clathrin-mediated endocytosis IONs uptake can also occur via clathrin-mediated endocytosis. It is known that some cells (such as macrophages) express clathrin, and clathrin forms clusters at these I. General Introduction 24 NPs capture areas. Clathrin assembly triggers the generation of coated pits of approximately 150 nm. Internalization occurs when the clathrin coating on the plasma membrane creates invaginations in the membrane, leading to clathrin-coated vesicles budding. Following internalization, the clathrin coating is recycled, leading to the fusion of the vesicle with an endosome where the sorting (which is mediated by microtubules) and dissociation of potential receptors takes place. Thereafter, degradation in lysosomes takes place and further transportation to the final destination occurs [142, 163, 164]. Lunov et al [165], having detected by transmission electron microscopy (TEM) that SPIONs and USPIONs are incorporated inside vesicles of approximately 100 nm in diameter, used monodansyl cadaverine, which is a specific inhibitor of clathrin-mediated endocytosis, and demonstrated that macrophages capture the carboxydextran-coated NPs though a clathrin-mediated endocytosis mechanism. Yang et al [166] using phenylarsine oxide (another inhibitor), reached a similar conclusion with Ferucarbotran. Likewise, Ayala et al [66] using dansylcadaverine also showed that carboxymethylsubstituted dextran-coated IONs were internalized using this mechanism. Internalization by this mechanism was attributed in this last case to the formation of a protein corona surrounding NPs. This protein corona is formed through non-specific interactions between serum proteins and NPs that are usually internalized via clathrinmediated and caveolae-mediated endocytosis [66]. 7.2.4. Caveolae-mediated endocytosis Caveolae (of diameter 50–100 nm) are invaginated, flask-shaped plasma membrane domains which are present in diverse cell types but particularly abundant in endothelial cells. Their structure is sustained by caveolins, which are a family of cholesterol-binding proteins. In addition to caveolin, caveolae are enriched in sphingomyelin, cholesterol and glycosphingolipids [147, 167]. Prijic et al [168] suggest that SPIONs uptake by mouse L929 fibroblasts cells, human melanoma SK-MEL-28 cells and human mesothelial MeT-5A cells occurs by caveolae-mediated endocytosis. Moreover, DMSA-coated SPIONs uptake in RAW264.7 cells was also reported to be caveolae-mediated [142]. Likewise, Ayala et al [66] using fillipin (which is a specific inhibitor of caveolae-mediated endocytosis), showed that carboxymethylsubstituted dextran-coated IONs were also internalized using this mechanism for the reason outlined above in 7.2.3. I. General Introduction 25 7.2.5. Receptor-mediated endocytosis 7.2.5.1. Transferrin receptor It has been described that the transferrin receptor is the most important route for iron transport through the capillary endothelium luminal membrane. It is possible that iron passes through the luminal membrane and in the form of Fe2+, enters the interstitial fluid [169]. Chen et al [170] hypothesized that gambogic acid loaded on magnetic IONs permeated the cell membrane through binding to the transferrin-binding site of transferrin receptor and endocytosis, which were potential routes for IONs uptake into cells. 7.2.5.2. Scavenger receptor (SR)-A Among the several receptor types involved in the endocytic process, macrophage SRs are an attractive family which has been thoroughly studied. These receptors were firstly identified as macrophage receptors involved in the recognition of modified lipoproteins. Presently, they comprise a family of eight different pattern recognition receptor subclasses, binding to a diversity of ligands, namely modified low density lipoproteins (LDL), polysaccharides, proteins, RNA and environmental particles, while playing a fundamental role in immunity and host defense [165, 171]. Under physiological conditions, SRs scavenge or clean up cellular debris as well as other similar materials and exert an important role in host defense. Under pathological conditions, SRs intervene in the recruitment, activation and transformation of several cells which may be correlated with the development of atherosclerosis and other disorders provoked by accumulation of the denatured materials, such as Alzheimer disease or multiple sclerosis [172]. It has been described that SPIONs uptake is mediated by SR-A [122, 173]. Raynal et al [173] used the SR-A inhibitors fucoidan and polyinosinic acid in competition experiments and observed that there was a dose-related inhibition of ferumoxides uptake, which clearly demonstrates that a SR-A-mediated endocytic pathway is implicated in IONs uptake by macrophages. SPIONs uptake into hepatocytes was also shown to be independent of caveolae, dynamin and clathrin-coated pits and mediated principally by a SR other than SR-A [174]. 7.2.5.3. Mac-1 receptor Another receptor possibly implicated in NPs capture is Mac-1 [175]. Both SPIONs and USPIONs are eliminated from the circulation by mononuclear phagocyte system cells I. General Introduction 26 CD4 molecules expressed on CD4+ lymphocytes were reported to be highly efficient and specific in CD4+ lymphocytes separation from whole blood, which can be applied in diverse biomedical applications namely diagnosis, treatment and monitoring of illnesses [226]. 10.2. Tissue repair IONs can be used to promote tissue repair. In fact, they were described (in the presence of the nerve growth factor) to synergistically instigate neurite outgrowth and upregulate a neural specific marker protein [227]. The possibility of using magnetic NPs in osteoporosis treatment has also been reported [228, 229]. The ability of these NPs (principally maghemite) to instantaneously enhance bone density and give rise to a further increase in osteoblast functions at the disease site was underlined. In addition, the magnetic properties of these NPs can be applied to guide drug molecules that were previously attached to the NPs under a magnetic field to targeted bone sites [228, 229]. 10.3. Drug delivery Magnetic NPs were used for the first time as delivery systems for drug delivery in cancer treatment in the late 1970s [168]. The magnetic properties of SPIONs permit their mechanic manipulation by a magnetic field gradient. This capacity of “acting at a distance” in combination with the intrinsic capacity that magnetic fields have to penetrate into human tissue, increases the specificity and selectivity of the therapy, enabling these particles to be conducted or held to a certain place through a magnetic field and heated to induce drug release or generate tissue hyperthermia [123, 230]. Therefore, the main benefits sought through use of nanovectors over simple drugs are: protection of the drug from their natural inhibitors, such as enzymatic degradation; specific and effective delivery of large quantities of therapeutic drugs utilizing biorecognition targets; pharmacokinetic and drug tissue distribution profile control; increased drug absorption into a selected tissue via increased retention effect and permeability; amelioration of intracellular penetration; avoidance of damage to healthy cells when cancer cells are killed and; low toxicity and immunogenicity [231, 232, 233]. SPIONs can work as vehicles for several molecules such as alkylating agents, plant alkaloids, antitumor antibiotics, antimetabolites, cytokines and monoclonal antibodies. Therefore, they function as delivery systems for therapeutic purposes [137, 168, 169]. A potential application is the conjugation of thrombin to IONs for use in the I. General Introduction 33 promotion of wound healing, given that thrombin is necessary for the conversion of fibrinogen to fibrin, whose action is critical and necessary for the early and late stages of wound healing [234]. IONs can be used to solve the problem of lack of sensitivity of tumor cells to cytotoxic drugs, which occurs in tumor treatment. Indeed, some reports have proven the synergistic effect of IONs on the delivery of anticancer drugs [5, 170, 235, 236], namely increasing the accumulation of the drug daunorubicin in leukemia cells. This accumulation could be related to the capacity of the IONs to block glycoprotein P function [237]. Previous investigation has demonstrated that daunorubicin and 5-bromotetrandrine attached to magnetic IONs revealed a considerable cytotoxicity effect on drug resistant leukemia K562/A02 cells [238]. SPIONs and USPIONs can be further modified by incorporation of a photosensitizer that has proven capable of generating singlet oxygen (1O2), useful in cancer therapy [239]. To achieve drug-targeting, SPIONs are frequently combined with targeting ligands such as antibodies, small molecules or peptides. This will target them for specific sites and allow rapid accumulation of SPIONs as well as the drugs they carry into the regions of interest. Some examples are folic acid, given that the folate receptor is usually overexpressed solely in cancer cells, namely in ovary, breast, kidney, colon and lung, and restricted in normal tissues [4, 97, 98]. Antisense oligonucleotides coupled with silicacoated magnetic IONs are also described as being a significantly attractive targeting delivery system for selective tumor targeting and specific cellular uptake [240]. Recently, Seabra et al [241] reported that IONs coated with DMSA or mercaptosuccinic acid may be nitrosated in their thiol groups. This formation of S-nitroso groups on the surface of IONs permits the release of nitric oxide, which can be useful in anticancer therapies. 10.4. Hyperthermia Hyperthermia is a therapeutic practice that relies on the enhancement of temperature in body tissues with the aim of changing the functionality of the cellular structures [86]. The earliest use of magnetic hyperthermia on tumors was described in 1957 by Gilchrist et al [242]. This author showed with in vitro assays that 5 mg of 20–100 nm diameter Fe2O3 NPs in lymph nodes (47 mg of Fe2O3 per gram of tissue) were able to trigger a temperature enhancement of 14 ºC in an alternating magnetic field of 200–240 Oe at 1.2 MHz [242]. However, the first prospective study for biomedical purposes in humans was described only in 1993. In 2010, magnetic hyperthermia trials involving the use of magnetic NPs passed preclinical stages and obtained regulatory authorization as a new clinical therapy denominated thermotherapy [243]. Recently, many IONs have been I. General Introduction 34 developed to be used in hyperthermia, namely γ-Fe2O3 NPs coated with L-3,4dihydroxyphenylalanine (L-DOPA) and tetra-methylrhodamine-5/6-isothiocyanate [162]. In this process, heat is produced as a result of magnetic hysteresis loss. When NPs are submitted to a high frequency AC magnetic field, they become heated due to Neel or Brownian relaxation losses in single-domain particles, which results in localized destruction of the cells [9, 35]. This local enhancement in temperature may be applied in medical treatments to destroy tumor cells and this is favored by the fact that cancer cells are more sensitive to abrupt rises in temperature above 43ºC than normal cells [86, 244]. In fact, the increase in temperature alters the performance of many structural and enzymatic cellular proteins, triggering repair enzyme inhibition, as well as alterations in DNA synthesis and conformation, together with alterations in differentiation and cell growth, which may lead to apoptosis. Hyperthermia may also trigger alterations in cell membrane which leads to a fall in transmembrane transport and provokes a destabilization of its potential [86]. Exposure of tumor cells to moderately high temperatures renders them sensitive to radiation and chemotherapy and may diminish their viability, depending on the exposure time and temperature [245]. Apart from killing cancer cells with heat, a host immune response is induced. Nevertheless, conventional hyperthermia systems encompass treatment solely once or twice per week, performed at intervals greater than 48 h to avoid thermotolerance provoked by the expression of heat shock proteins [245]. In addition, it is difficult to heat the local tumor region to the desired temperature without provoking damage in the normal tissue [245]. However, hyperthermia using magnetite NPs has proven to be efficient in animals with different types of tumor, including T-9 rat glioma, B16 mouse melanoma, VX-7 squamous cell carcinoma in rabbit tongue, Os515 hamster osteosarcoma and MM46 mouse mammary carcinoma [245]. 10.5. Transfection Transfection or gene therapy implies the introduction of genetic materials namely small interference RNA, oligonucleotides, plasmid DNA, messenger RNA and doublestranded DNA into tissues or target cells to express knockdown mutated deleterious genes, heterogeneous gene products, and substitute with functional genes. Gene delivery has demonstrated great promise in genetic disease treatments such as thalassemia, cystic fibrosis, influenza A, Huntington’s disease, Parkinson’s disease, cancer, inherited color blindness, etc. The gene carrier (usually denominated as a vector) is required to transfer the genetic sequences for gene expression into the cellular compartment. Vectors can be generally classified into two groups: nonviral and viral [103, 246, 247]. I. General Introduction 35 Magnetofection is a process whereby magnetic NPs linked to DNA are, under the influence of an external magnetic field, transfected into cells [9, 13]. NPs present numerous advantages as gene vectors over viral carriers, namely they: - do not induce immune responses, since they are not biological materials; - have limited genotoxicity and cytotoxicity; - are able to mediate the introduction of foreign genes into the DNA of host chromosomes and consequently, foreign genes are expressed stably; - have the ability to protect foreign genes from host enzymes and complement; - may kill or destroy some viruses [248]. NPs cannot effectively label the required cells because of the repulsive electric interaction between the NPs and cell membrane, given that both the SPIONs and cell membrane possess a negatively charged surface. Therefore, a complex composed of SPIONs and transfection agents is frequently used to increase its labeling efficiency. The use of transfection agents is beneficial in transferring SPIONs to the target cells. The commonly used transfection agents such as PLL, lipofectamine, PEI and protamine sulphate are predominantly cationic [87, 103, 249, 250]. Positively charged magnetic NPs are endocytozed in the tight fitting vesicles due to the electrostatic interaction between positive charged magnetic NPs and the anionic cell membrane. Subsequently, the unsaturated amino groups trap the pumped-in-protons by the ATPase located on the endosomal membrane, which conducts to Clions retention and water molecules in the endosome. When water retention accumulates, swelling and bursting of the endosomes occur which results in the genetic material and magnetic NPs being released into the cytoplasm [103]. Therefore, NPs have the potential to be used in magnetofection. It has been reported that CLIO-Tat may be used to transfect CD34q hematopoietic T cell precursors and offers a suitable combination for effective T lymphocytes loading [251]. CLIO which show polyethylene glycol moiety and covalently linked branched PEI, mediates efficient and rapid transfection in primary vascular human umbilical vein endothelial cells (HUVEC) and effectively inhibits plasminogen activator inhibitor-1 expression which is responsible for several vascular dysfunctions, such as atherosclerosis and vascular inflammation. For this reason, these IONs offer a possible approach to transfect highly sensitive HUVEC [252]. I. General Introduction 36 10.6. DNA detection IONs readily interact with proteins and as such, it is possible to combine several hydrophilic macromolecules (namely peptides and nucleotides) with IONs, which could be applied for highly sensitive detection of the DNA sequence [253, 254]. 10.7. Tissue soldering SPIONs may be used for tissue soldering [108]. For this purpose, their specific size requires an assessment for optimized heating ability given that these SPIONs may be “tuned” with their corresponding electromagnetic frequency, also called Neél and Brownian relaxation [108, 255]. 10.8. Biofilm treatment and antibacterial activity IONs may provide some supplementary advantages for biofilm treatment since iron restriction is intimately connected to the physiological onset of biofilm formation for common pathogenic bacteria, namely Staphylococcus aureus, S. epidermidis and Pseudomonas aeruginosa [229]. Inhibition of a bacterial colony formation even when the SPIONs concentration is 10 µg/mL has been described [229]. In vitro antimicrobial activity of magnetic poly(γ-glutamic acid)-coated NPs was also reported against S. enteritidis, E. coli and S. aureus [67]. It was also illustrated that IONs may be able to enhance the antibacterial activity of the bacitracin, which results in a lower dosage with a subsequent reduction in side effects associated with the antibiotics [256]. 10.9. Vaccine carriers The potential of IONs as potential vaccine carriers has also been reported. However, further work is being carried out to induce antibody responses in vivo [257]. Hu et al reported an ocular mucosal administration of IONs containing DNA vaccine pRSCgD-IL-21 as promising in the inhibition of herpes stromal keratitis due to the efficient transport of plasmid DNA vaccine into target cells [258]. More recently, it was reported that IONs could be linked to polyinosinic-polycytidylic acid, an activator of Toll-like receptors 3 which is described as being a promising adjuvant for vaccines. Given that this receptor is expressed by tumor cells of several cancers, this system could prove an alternative in cancer treatment [259]. I. General Introduction 37 10.10. Peroxidase activity Magnetic IONs were reported to have peroxidase behavior which is an attractive property apart from their superparamagnetism. Since IONs can compete with natural enzymes due to their robustness, ease of preparation and stability in adverse conditions, they may constitute robust, simple, easy-to-make and cost-effective biosensors in the future. For example, the capacity to catalyze the organic substrates oxidation to generate a color change and/or to diminish their toxicity is regularly employed as a detection tool or in the treatment of wastewater [260, 261, 262]. However, one of the most usual peroxidase-relevant applications is related to the oxidation reaction by hydrogen peroxide. Glucose detection is frequent in the laboratory or in clinics and is a common application of peroxidase. IONs also exhibit high sensitivity in glucose detection particles [261]. 11. Final notes IONs (USPIONs, <50 nm and SPIONs, >50 nm) encompass electrical, thermal, mechanical and imaging properties that gain relevance regarding their use in biomedicine. Due to these properties, several commercial formulations of these NPs (namely Ferumoxides, Ferucarbotran, Ferumoxtran-10, among others) have been extensively used in MRI. Tissue repair, drug delivery, hyperthermia and transfection among other biomedical applications mentioned in this review also represent other applications of this type of NPs. The number of applications of IONs is growing exponentially and this growth is estimated to increase endlessly in the future. New types of IONs are coming to market and significant research is being directed towards developing new types of IONs with different sizes and coatings for innovative applications in biomedicine. However, and despite the importance and enormous utility of these NPs in biomedicine, it will be important in the near future to evaluate their safety and the way in which the different types of existing and prospective IONs interact with the human body. Equally important will be the need to create a toxicology database for better control of the less beneficial effects of these NPs. I. General Introduction 38 12. List of Abbreviations 1O2 – Singlet oxygen BBB – Blood-brain barrier C2A-GST – Domain of synaptotagmin I; glutathione S-transferase CLIO – Cross-linked iron oxide nanoparticles CNS – Central nervous system DMSA – Dimercaptosuccinic acid DNA – Deoxyribonucleic acid FDA – Food and Drug Administration HUVEC – Human umbilical vein endothelial cells IONs – Iron oxide nanoparticles LDL – Low density lipoproteins L-DOPA – L-3,4-Dihydroxyphenylalanine MIONs – Monocrystalline iron oxide nanoparticles MRI – Magnetic resonance imaging MSC – Mesenchymal stem cells NPs – Nanoparticles PAA – Polyacrylic acid PEG – Polyethylene glycol PEI – Polyethylenimine PEO – Polyethylene oxide PGMA – Poly(glycidyl methacrylate) PLGA – Poly(lactic-co-glycolic acid) I. General Introduction 39 PLL – Poly(L-lysine) PTA – Polycationic transfection agents PVA – Polyvinyl alcohol PVP – Poly(vinylpyrrolidone) RES – Reticuloendothelial system ROS – Reactive oxygen species SPIONs – Superparamagnetic iron oxide nanoparticles SR – Scavenger receptors TEM – Transmission electron microscopy USPIONs – Ultra-small superparamagnetic iron oxide nanoparticles TEM – Transmission electron microscopy 13. Conflicts of Interest The authors declare that there are no conflicts of interest. 14. Acknowledgements Diana Couto and Marisa Freitas acknowledge the FCT financial support for the PhD and Pos-doc grants (SFRH/BD/72856/2010 and SFRH/BPD/76909/2011, respectively), in the ambit of “POPH - QREN - Tipologia 4.1 - Formação Avançada” cosponsored by FSE and national funds of MCTES. 15. References [1] Azevedo, R.B.; Valois, C.R.; Chaves, S.B.; Silva, J.R.; Garcia, M.P. Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles. 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General Introduction 58 I.1.2.1. MECHANISMS OF CYTOTOXICITY The effects of IONs on the viability and/or cytotoxicity of several cells are variable according to the type of IONs, coating, concentration, interaction with different types of cells, and have been described in several studies. The mechanisms behind the IONs cytotoxicity are not completely established and various mechanisms are proposed (figure 1), as discussed in the following chapter. Figure 1. Possible mechanisms underlying the cytotoxic effects of IONs (ATP: Adenosine triphosphate; Cyt c: Cytochrome c; IONs: Iron oxide nanoparticles; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor α). I. General Introduction 65 I.1.2.1.1. REACTIVE OXYGEN SPECIES, REACTIVE NITROGEN SPECIES AND PEROXIDASE ACTIVITY ROS are defined as molecules containing one or more oxygen atoms, which are more reactive than molecular oxygen. RNS, mainly peroxynitrite anion (ONOO-), nitrogen dioxide, and its sub-products, derive from reactions between superoxide radical (O2•-) and nitric oxide radical (•NO) [22]. It has been reported that the potential mechanism of IONs cytotoxicity involves the formation of ROS such as hydrogen peroxide (H2O2), hydroxyl radical (HO•), hydroperoxyl radical (HO2•), and O2•-, among others. When produced in abundance, ROS can lead to oxidative stress, given that they disturb the balance between oxidative pressure and antioxidant defense, which results in damage to biomembranes [20] (figure 1). As a good example, exposure of PC12 cells to Fe3O4 NPs was shown to induce a significant increase of intracellular ROS, which was associated to the release of cytochrome c from mitochondria, resulting in initiation of the intrinsic apoptosis pathway [23]. In another study, the free radical scavenger edaravone, abolished caspase 3 activation and apoptosis in Kupffer cells of in mice treated with Resovist® [carboxydextran-coated superparamagnetic IONs (SPIONs)] [24]. After internalization, IONs are presumably degraded into iron ions within the lysosomes by hydrolysing enzymes, at low pH. Free iron ions can potentially cross the nuclear or mitochondrial membrane and induce redox cycling and catalytic chemistry via Fenton reaction [H2O2 + Fe2+ → Fe3+ + HO− + HO•], the most prevalent source of ROS in biological systems, and thus catalyze the formation of the highly reactive HO• that damages cellular membranes, depolymerizes polysaccharides, causes deoxyribonucleic acid (DNA) strand breaks, inactivates enzymes, and initiates lipid peroxidation [15,20,25]. In Fe3O4 NPs, iron is present as a mixture of Fe3+ and Fe2+ ions, and it is possible that surface Fe2+ ions contribute to the Fenton reaction. On the other hand, in Fe2O3 NPs, iron ions are mostly in the ferric state; thus maghemite would produce less radicals [26]. IONs are also known to be phagocytosed by neutrophils and other phagocytes. Upon formation of the phagolysosome nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are activated, generating O2•- and this generation of O2•- would lead to the production of other ROS. It is also possible that NPs-induced damage of the mitochondria could determine Ca2+ release into the cytosol, where Ca2+-dependent enzymes, such as nitric oxide synthase, become activated, resulting in production of •NO and ONOO- [27,28] I. General Introduction 66 (figure 2). Depending on the type of IONs used, the time at which maximal ROS and RNS levels are generated can vary greatly, from approximately 4 h for citrate-coated IONs to 24–32 h for dextran-coated ones [29]. Figure 2. IONs-induced production of ROS and RNS (H2O2: Hydrogen peroxide; HO•: Hydroxyl radical; HOCl: Hypochlorous acid; iNOS: Inducible nitric oxide synthase; IONs: Iron oxide nanoparticles; NADPH: Nicotinamide adenine dinucleotide phosphate; •NO: Nitric oxide radical; NOS: Nitric oxide synthase; 1O2: Singlet oxygen; O2•-: Superoxide radical; ONOO-: Peroxynitrite anion). There are several studies performed to evaluate the effect of different types of IONs on ROS and RNS production in different types of cells and results indicate that the effect on these cells depends on the different characteristics of the IONs (namely coating, concentration and type of IONs) and the different interaction of these IONs with the different cells and their receptors. For example, Fe2O3 NPs was reported to increase ROS production and inhibit catalase in Hep-2 human epithelial cells [30], while FeRex® I. General Introduction 67 (dextran-coated SPIONs) had no effects on ROS production in bone marrow dendritic cells [31]. In these circumstances, detoxifying enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase can be produced in order to prevent ROSinduced damage and the extent of ROS production and consequent damage may be dependent on the production of these enzymes, whose expression and activities vary from cell to cell [27]. The techniques used to assess ROS and RNS production may be responsible for the different results reported in the literature. Fe3O4 NPs was shown to increase ROS production in human lung cancer A549 cells [32,33], while Guadagnini et al [34] reported that the same IONs did not induce ROS production in the same cell line. The fact that Konczol et al [32] and Ahamed et al [33] presented contradictory results when compared to the results presented by Guadagnini et al [34] may be related to the different probes used: while Konczol et al [32] and Ahamed et al [33] used dichlorodihydrofluorescein diacetate, a probe that detects H2O2, HO•, •NO and ONOO-, Guadagnini et al [34] used hydroethidine, a probe that only detects O2•- [35]. IONs peroxidase-like activity has as well been described as one of the potential mechanisms involved in the cytotoxicity of IONs. In fact, IONs may catalyze H2O2 to react with various intracellular molecules including proteins, lipids, and nucleic acids through peroxidase-like activity, thus causing irreversible cell damage. On the contrary, when IONs have catalase-like activity, they would rather protect cells from oxidative stress through decomposition of H2O2 into H2O and O2. When IONs are located in lysosomes, the acidic environment contributes to their peroxidase-like activity rather than catalase-like activity [36]. I.1.2.1.2. DISRUPTION OF THE CYTOSKELETON IONs are described as being capable of affecting the actin cytoskeleton. The cytoskeleton is a dynamic network consisting of actin polymers, microtubules, and associated proteins. Actin, in particular, plays an important role in cell shape, adhesion, and motility. Increasing evidence shows that the actin cytoskeleton is essential to endocytotic processes, including pseudopod extension, phagocytotic engulfment, cell surface remodeling for vesicle formation and movement as well as to the maintenance of I. General Introduction 68 the cell morphology and their effect on signaling pathways monitoring several parameters such as cell death, migration and differentiation [37,38]. It is described that the formation of cytoplasmic vacuoles containing internalized IONs disrupts the cytoskeleton and cell membrane by either chemically or physically impeding actin microfilament formation [39]. In fact, Miller et al [40] suggested that NPs could interact with the extracellular domain of integrins (proteins that promote actin assembly) and thereby affect their activation and intracellular signalling cascade. It is reported that IONs affect the morphology and actin cytoskeleton and the formation and maturation of focal adhesion complexes in porcine aorthic endothelial cells [41], human blood outgrowth endothelial cells [42,43], as well as human mesenchymal stem cells [44]. Wu et al [45] reported that citrate-coated Fe3O4 NPs interfere with the polymerization and depolymetrization of the tubulin in human umbilical vein endothelial cells, and induce disruption of F-actin and microtubules, resulting in a disorganization of the cytoskeleton. Gupta et al [46] reported also a rapid disruption of actin and microtubule structures in human fibroblasts as well as Apopa et al [47] reported that Fe2O3 NPs induced the activation of protein kinase B (Akt) and inhibited glycogen synthase kinase-3β in a phosphoinositide-3-kinase dependent manner in human microascular endothelial cells, which results in microtubule remodeling. I.1.2.1.3. INTERFERENCE WITH ATP PRODUCTION IONs were also described as exerting a decrease in ATP production in mitochondria [48]. In fact, it is described that Fe2O3 NPs decreased the activity of Mg2+-ATPase in a dose-dependent manner in brains of rats, which may hamper the ATP synthesis and thus lead to mitochondrial disorganization [49]. The same authors also reported that Fe2O3 NPs decreased the activities of total, Na+-K+, and Ca2+-ATPases in a dose-dependent manner in brains of rats, possibly due to the interaction of these IONs with the enzymes and suggest that the observed decreased in the activity of Na+-K+-ATPases may cause alterations in the cell-membrane permeability, presumably resulting in impaired ionoregulatory activity as well as cause an imbalance in cellular functions, while the decrease in the activity of Ca2+-ATPases may conduct to disturbances in numerous cellular processes, such as exocytosis, cell proliferation, gene transcription, cell differentiation, synaptic maturation, neurotransmitter release, intracellular signaling, muscle contraction and cell survival [49]. I. General Introduction 69 I.1.2.1.4. PARAMETERS THAT AFFECT THE CYTOTOXICITY OF IRON OXIDE NANOPARTICLES I.1.2.1.4.1. CELL TYPE It is important to consider that IONs cytotoxicity depends on diverse parameters. One of them is the cell type. For example, Horie et al [50] reported that Fe2O3 NPs caused a decrease in the viability of human keratinocyte HaCaT cells, while for similar conditions and concentrations, Sun et al [51] reported that Fe2O3 NPs did not cause decrease in the viability of human cardiac microvascular endothelial cells. It is also possible to verify that certain cells are more propense to IONs toxicity. For example, in lymphocytes, there are many studies performed that indicate that IONs trigger a decrease in viability [52-54], while most of the authors that studied the effect of IONs on mesenchymal stem cells came to the conclusion that they do not exert cytotoxicity [55-59]. Although there is not any specific explanation for these facts, possible explanations could be the presence of distinct cell surface receptors in these cell types leading to differential downstream signalling events, discrepancy of NPs uptake, differences in the sensitivity of cellular organelles to IONs induced insult (lysosomal/mitochondrial damage), differential handling of NPs after uptake and different inherent capacity to deal with oxidative stress [34]. Noteworthy, for most of the cells, ROS production appeared to be directly related to the quantity of lysosomes formed following SPIONs exposure [60]. I.1.2.1.4.2. COATING OF IONs The coating used also influence cytotoxity of IONs. In fact, the interaction of the IONs coating with proteins may change their biological reactivities [50], although coatings with known biocompatible compounds do not always exclude an unexpected increase in cytotoxicity or pro-inflammatory response [34]. For example, oleate itself and non-coated Fe3O4 NPs were not found to be toxic to TK6 cells and lymphocytes, while oleate-coated Fe3O4 NPs were found to be cytotoxic to the same cellular lines [61]. Likewise, Zhu et al [62] reported that, while aminosilane, dextran and non-coated SPIONs did not resulted in I. General Introduction 70 decreased cell viability, silica-coated SPIONs decreased cell viability in RAW 264.7 cells. In fact, coordination of the coating agent with the nanostructure influences the entry into or interaction of both the nanostructure and surface chemicals with cells, thereby magnifying any interactions (positive or negative) with cellular components; a second, alternative explanation for this fact is the variance in the effectiveness of the coatings to shield the nanostructures from potentially adverse interactions with cellular components [39,63]. Shen et al [64] reported that the cytotoxicity of NPs with amine groups on their surface stems from the strong electrostatic interaction between the positively charged NPs and the negatively charged cell membranes. On the other hand, the absence of cytotoxic effects attributable to poly(L-lysine) (PLL)-Endorem® (dextran-coated SPIONs) could be explained by the delayed release of free iron into the intracellular environment due to the slower degradation of their double-coating [65]. In a similar way, Fan et al [66] reported that, while Fe3O4 NPs decreased cell viability in hepatic cell line LO2, O-carboxymethyl chitosan-coated Fe3O4 NPs, folic acid-carboxymethyl chitosan-coated Fe3O4 NPs and dextran-coated SPIONs did not trigger the same effect. In this case, coatings were able to reduce Fe3O4 NPs cytotoxicity. For example, it is known that O-carboxymethylchitosancoated Fe3O4 NPs have carboxyl groups that are bound to the surface of Fe3O4 by covalent bonds of “O” of the hydrous Fe3O4 NPs. These carboxyl groups will confer a negative charge to Fe3O4 NPs, which prevents agglomeration by the electrostatic repulsion. Moreover, this coating may increase the surface hydrophilicity, minimizing adherence to cell membranes by hydrophobic interaction, which minimizes phagocytosis [66]. I.1.2.1.4.3. TYPE OF IONs Several types of IONs can be synthetized, magnetite (Fe3O4), hematite (α-Fe2O3) and maghemite (γ-Fe2O3) being the most common [15,67]. The type of IONs may also influence their cytotoxicity. For example, Karlsson et al [68] described that Fe2O3 NPs caused a slight decrease in viability of A549 cells, while Fe3O4 NPs did not cause the same effect. However, Park et al [69] reported that, in a MH-S cell line, Fe3O4 NPs caused a greater cell viability decrease than Fe2O3 NPs. Likewise, Chen et al [36] reported that, while 2,3-dimercaptosuccinic acid (DMSA)-coated γ-Fe2O3 NPs are not cytotoxic to human neuronal glioblastoma U251 cells, DMSA-coated Fe3O4 NPs decreased cell I. General Introduction 71 viability at a lower concentration in the same cellular line. In Fe3O4 NPs, iron is present in the form of Fe3+ and Fe2+ ions, while in Fe2O3 NPs iron is mostly in the form of Fe3+ ions [26]. In fact, Fe2+ is released into the solution much faster than Fe3+ because the bonds between the reduced iron and O2− ions of the crystalline lattice are weakened [70]. However, materials only containing Fe3+ at the surface, such in the case of Fe2O3 NPs, may also release HO• through a Fenton-like reaction that requires previous reduction of Fe3+ to Fe2+ by endogenous molecules such as ascorbic acid or by reaction of Fe3+ and H2O2 with HO2• as the propagating intermediate, which may increase cytotoxicity [70,71]. I.1.2.1.4.4. SOLUBILITY OF IONs The solubility of IONs has recently been shown to directly affect its cytotoxicity [72]. It is known that the low pH of endosomes/lysosomes can solubilize the iron core within a few days, releasing ferric iron Fe3+ into the cytoplasm and consequently the cytotoxicity may increase [73]. However, IONs of extremely low solubility may also be cytotoxic because they will be persistent within the biological system and may provoke a range of long-term effects involving carcinogenic, mutagenic, or teratogenic influence in the organism [72]. I.1.2.1.4.5. SURFACE CHARGE OF IONs The surface charge of IONs influences their stability as well as the cellular internalization and trafficking pathways, therefore influencing their cytotoxicity [9,74]. It is generally accepted that the NP-protein “corona”, a dynamic layer of proteins and other biomolecules that adsorbs to NP surfaces immediately upon contact with living systems, mediates the interaction of IONs with the cellular machinery [18]. Generally, the surface charges of the IONs are negative due to their interaction with the hydroxyl groups of the water molecules, and these surface charges originate an electric field that attracts counterions [75]. IONs, due to their strong van der Waals forces, have a significant interaction with the surrounding environment, which alters the physical properties of the NPs, depending upon the presence of ionic (salt) and biomolecules in their vicinity, gravitation, diffusion, convection forces, cell type, pH and protein composition, either in I. General Introduction 72 culture media or in body fluids, and the extent of agglomeration [76,77]. Proteins such as albumin, immunoglobulin, fibrinogen, and apolipoproteins were found to play an important role in the agglomeration of IONs [78]. Simberg et al [79] reported that IONs have the ability to bind to mouse plasma proteins, namely histidine–proline rich glycoprotein, high molecular weight kininogen, plasma prekallikrein, mannose-binding lectins (MBL), mannose-binding lectin-associated serine proteases, apolipoproteins, beta-2 glycoprotein, clotting factors coagulation factor XI (FXI) and coagulation factor XII (FXII), hemoglobin, hemoglobin-binding hemopexin (proteoglycan-4), vinculin, tubulin-1 and talin-1. The fact that kallikrein, kininogen, FXI and FXII can be bound to IONs surface may potentially initiate the activation of the intrinsic pathway of blood clotting [79]. IONs have also been described as causing disruption of microvilli, through disruption of the adherens junctions [21,80]. In fact, protein fibrillation may occur due to protein-NPs interactions [18]. It was also found that interaction of Fe3O4 NPs with amyloid aggregates led to a decrease of the amount of amyloids by depolymerization of amyloid structures and inhibition of lysozyme aggregation [81]. IONs can also disturb the electronic and/or ionic transport chains due to the strong affinity of the NPs for the cell membrane [82]. Therefore, the immediate environment of the NPs (presence of biomolecules, chemical composition, and physiological properties of the liquid in which they are suspended) may modulate their toxicity on the basis of agglomeration, that may depend on the surface chemistry and charge of IONs [77]. It is reported that IONs do not need to enter into the cells to exert cytotoxicity. In the case of non-coated IONs, as they have a negative charge and the cell membrane also has a negative charge, there is a slight electrostatic repulsion between cells and the noncoated-IONs, which influence the interaction between cells and IONs. However, cell membrane receptors have different aminoacid compositions that comprise negative, neutral, and positive charges; therefore, the effect of charge on the interaction between transmembrane receptors or channels and IONs continuously changes according to the type of IONs, coating, surface charge, among many other previously mentioned factors [48,61]. The different charge might also influence the interactions with other compounds present in medium, such as serum proteins, as well as with cell membranes, and thus could have impact on internalization of NPs and toxicity [61]. I. General Introduction 73 I.1.2.1.4.6. SIZE OF IONs IONs size is also a parameter that influences their cytotoxicity. Ying et al [52] reported that 50 nm IONs were more toxic than 10 nm IONs to A3 human T lymphocytes. Likewise, Faust et al [83] reported that 78 nm α-Fe2O3 NPs are more toxic to BeWo b30 plancentary epithelial cell line than 50 nm α-Fe2O3 NPs, the 15 nm α-Fe2O3 NPs being not toxic to the same cellular line. Larger particles possess larger effective interaction area with more functional groups for accessing to the cell, in comparison with smaller NPs. Within this specific area, there are more functional groups on the individual large particle [52,84]. Larger aggregates, as it occurs in the case of non-coated IONs, are considered more cytotoxic, as they may mechanically damage the cell and also deform the nucleus on the entry into the cell [61,85]. However, it is also reported that smaller NPs are more toxic. This is, probably, due to their large specific surface area and hence larger surface reactivity toward biological systems [86]. According to this theory, Zhang et al [21] reported that α-Fe2O3 NPs with 26 nm were more cytotoxic to CaCo-2 cells than α-Fe2O3 NPs with 53, 76 and 98 nm. Also, size may influence the uptake of IONs by phagocytic cells. In fact, IONs with dimensions superior to 50 nm are usually phagocytized by cells of the reticuloendothelial system in liver and spleen, being eliminated in a short period of time, while IONs with dimensions inferior to 50 nm are able to move through the capillary wall and be phagocytozed by phagocytic cells that are also in other organs, namely bone marrow and lymph nodes [87,88]. However, when IONs are smaller than 10 nm, they may be eliminated via extravasation and renal clearance [15]. I.1.2.1.4.7. EXPERIMENTAL CONDITIONS Some erroneous factors derived from the experimental conditions have to be taken into account. The adsorption of the components of the culture media onto IONs induces a starvation state of cells in vitro. Thus, the adsorption effect that occurs when cytotoxicity experiments are performed may influence their toxicity in vitro [50]. These effects of adsorption are expected to be proeminent when the NPs are smaller, given that the adsorption ability of NP increases as the particle size decreases [50]. I. General Introduction 74 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Human bone marrow mesenchymal stromal cells PLL-coated γ-Fe 2 O 3 NPs (15.4 μg/mL), mannose-coated γ-Fe2O3 NPs (15.4 μg/mL), PLL-coated Endorem® (15.4 μg/mL), Endorem® (15.4 μg/mL) and γ-Fe2O3 NPs (15.4 μg/mL) Decrease in cell viability [65] Human amniotic membrane-derived mesenchymal stem cells Resovist® (28 μg/mL) Decrease in cell viability and loss of morphology [144] Mouse mesenchymal stem cells Protamine-coated Feridex® (25 μg/mL) No evidence of cytotoxicity [57] Mouse mesenchymal stem cells Poly(lactide-co-glycolide)-coated Fe3O4 NPs (1 mM) and cellulosecoated Fe3O4 NPs (1 mM) No evidence of cytotoxicity [58] Rat mesenchymal stem cells PEI-polyacrylic acid (PAA)-coated Fe3O4 NPs (100 μg/mL) No evidence of cytotoxicity [59] Pulmonary cells Human lung epithelial cells BEAS-2B Tetramethylammonium hydroxidecoated Fe3O4 NPs (6.0 x 1012 NP/mL) No evidence of cytotoxicity [89] Human lung epithelial cells BEAS-2B α-Fe 2 O 3 NPs (50 μg/mL) Decrease in cell viability [77] Human lung epithelial cells BEAS-2B Fe 2 O 3 NPs (50 μg/cm2) Decrease in cell viability [145] Human lung cancer A549 cells Fe 2 O 3 NPs (80 μg/mL) Decrease in cell viability [68] Human lung cancer A549 cells Fe 3 O 4 NPs (80 μg/mL) No evidence of cytotoxicity [68] Human lung cancer A549 cells Poly (ethylene oxide)-block-poly(γmethacryloxypropyl trimethoxysilane)- coated IONs (0.5 mg/mL) No evidence of cytotoxicity [146] Human lung cancer A549 cells α-Fe 2 O 3 NPs (100 μg/cm2) No evidence of cytotoxicity [70] Human lung cancer A549 cells Fe 2 O 3 NPs (10 mg/mL) Decrease in cell viability [50] Human lung cancer A549 cells Fe 3 O 4 NPs (0.5 μg/mL) Decrease in cell viability [147] Human lung cancer A549 cells Fe 3 O 4 NPs (200 μg/mL) Decrease in cell viability [66] Human lung cancer A549 cells Carboxyethylsilanetriol-coated SPIONs (114 μg/mL) Decrease in cell viability [60] Human lung cancer A549 cells IONs (25 μg/mL) Decrease in cell viability and loss of mitochondrial membrane potential [148] Human lung cancer A549 cells Fe 3 O 4 NPs (25 μg/mL) Decrease in cell viability [33] Human lung cancer A549 cells Sodium oleate-coated Fe 3 O 4 NPs (3 μg/cm2) and Fe3O4 NPs (37.5 μg/cm2) Decrease in cell viability [34] Human lung cancer A549 cells Sodium oleate-coated Fe 3 O 4 NPs (0.6 mM), sodium oleate and PEG-coated Fe3O4 NPs (0.35 mM), sodium oleate, PEG and poly(lactide-co-glycolic acid)- coated Fe3O4 NPs (0.1 mM) Decrease in cell viability [149] Human lung cancer A549 cells Fe 3 O 4 NPs (200 μg/cm2) Slight loss of mitochondrial membrane potential [32] I. General Introduction 81 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Human lung fibroblast IMR-90 cells IONs (100 μg/mL) No evidence of cytotoxicity [148] Human lung fibroblast IMR-90 cells Fe 2 O 3 NPs (10 μg/cm2) Decrease in cell viability [145] Rat BALF Fe 3 O 4 NPs (aerosol concentration of 640 mg/m3 for 4 hours) Decrease in cell viability [95] Rat BALF γ-Fe 2 O 3 NPs (90 μg/m3 for 6 hours during 3 days) No evidence of cytotoxicity [150] Rat BALF γ-Fe 2 O 3 NPs (57 µg/m3 for 6 hours during 3 days) No evidence of cytotoxicity [151] Mouse lung alveolar type II (C10) epithelial cells Carboxy-coated Fe 3 O 4 NPs (10 µg/mL) Decrease in cell viability [152] Mouse lung alveolar type II (C10) epithelial cells Amine-coated Fe 3 O 4 NPs (200 µg/mL) No evidence of cytotoxicity [152] Human lung embryonic HEL 12469 cells Sodium oleate-coated Fe 3 O 4 NPs (0.6 mM), sodium oleate and PEG-coated Fe3O4 NPs (0.35 mM), sodium oleate, PEG and poly(lactide-co-glycolic acid)- coated Fe3O4 NPs (0.1 mM) Decrease in cell viability [149] Human bronchial 16HBE epithelial cells Sodium oleate-coated Fe 3 O 4 NPs (7.5 μg/cm2) and Fe3O4 NPs (15 μg/cm2) Decrease in cell viability [34] Human lung adenocarcinoma ASTC-a1 PLL-coated SPIONs (50 μg/mL) Decrease in cell viability [153] Lewis lung carcinoma cell line Poly(3-(Trimethoxysilyl)propyl methacrylate -r-PEG monomethyl ether methacylate)-coated Fe3O4 NPs (100 μg/105 cells) No evidence of cytotoxicity [154] Rat lung Fe 3 O 4 NPs (500 µg/mL) No alteration of mitochondrial respiratory chain complexes I, II, III and IV activities [139] Osteoblast cells Human osteosarcoma (U2OS) Carbon-coated IONs (100 μg/mL) Decrease in cell viability [131] Brain cells Human neuronal glioblastoma U251 cells DMSA-coated γ-Fe 2 O 3 NPs (100 μg/mL) No evidence of cytotoxicity [36] Human neuronal glioblastoma U251 cells DMSA-coated Fe 3 O 4 NPs (25 μg/mL) Decrease in cell viability [36] Human neuronal glioblastoma U251 cells Tetramethylammonium 11aminoundecanoate-coated Fe3O4 NPs (100 μg/mL) Decrease in cell viability [82] Human neuronal glioblastoma U251 cells Protamine sulfate-coated Feridex IV® (100 μg/mL) No evidence of cytotoxicity [155] Human primary glioblastoma cell line U87 Tetramethylammonium 11aminoundecanoate-coated Fe3O4 NPs (100 μg/mL) No evidence of cytotoxicity [82] I. General Introduction 82 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects U87MG glioblastoma cells USPIONs (0.03 μmol/mL) and arginine-glycine-aspartic acid-coated USPIONs (0.03 μmol/mL) No evidence of cytotoxicity [156] U87MG glioblastoma cells Fe 3 O 4 NPs (25 µg/mL) Protection from H 2 O 2 -induced cytotoxicity [157] Human neural stem cell line HB1F3 Feridex IV® (25 μg/mL), MION-47(25 μg/mL) , CLIO-NH2 (25 μg/mL) and tatCLIO (25 μg/mL) No evidence of cytotoxicity [158] Neuro-2A mouse neuroblastoma Fe 3 O 4 NPs (200 μg/mL) No evidence of cytotoxicity [159] Human glioma cell line D54MG Tetramethylammonium 11aminoundecanoate-coated Fe3O4 NPs (100 μg/mL) Decrease in cell viability [82] Human brain tumor cell G9T/VGH Tetramethylammonium 11aminoundecanoate-coated Fe3O4 NPs (100 μg/mL) Decrease in cell viability [82] Glioma cell line SF126 Tetramethylammonium 11aminoundecanoate-coated Fe3O4 NPs (100 μg/mL) No evidence of cytotoxicity [82] Human isolated from brain epithelium U373 Tetramethylammonium 11aminoundecanoate-coated Fe3O4 NPs (100 μg/mL) No evidence of cytotoxicity [82] Oligodendroglia OLN-93 cells DMSA-coated IONs (4 mM) Loss of the initial bipolar shape and bright intracellular vesicles [160] Oligodendroglial OLN-93 cells DMSA-coated IONs (1000 μM) No evidence of cytotoxicity [161] BV2 microglial cells α-Fe 2 O 3 NPs (0.2 mmol/L) and γFe2O3 NPs (0.2 mmol/L) Large number of cellular vesicles, proliferation of lysosome, swelling of endoplasmic reticulum and disappearance of mitochondrial cristae [162] Human brain-derived endothelial cells Amino polyvinyl alcohol (PVA)-coated SPIONs (200 μg/mL) and Fe3O4 NPs (200 μg/mL) No evidence of cytotoxicity [163] Human brain-derived endothelial cells Oleic acid-coated Fe 3 O 4 NPs (150 μg/mL) Decrease in cell viability [163] C17.2 neural progenitor cells Resovist® (150 µg/mL) and Endorem® (200 µg/mL) No evidence of cytotoxicity [43] Chick cortical neurons FluidMAG-Amine (aminosilane-coated Fe3O4 NPs (100 μg/mL) No evidence of cytotoxicity [19] Chick cortical neurons FluidMAG-D (dextran-coated Fe 3 O 4 NPs) (50 μg/mL) Decrease in cell viability [19] I. General Introduction 83 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Chick cortical neurons FluidMAG-PEA (poly- (dimethylamine-co-epichlorhydrin-coethylendiamine)-coated Fe3O4 NPs) (10 μg/mL) Decrease in cell viability due to remotion of plasma membrane of the neurons [19] PC12 cells Citrate-coated very small SPIONs (VSOP) (400 μg/mL) Decrease in the formation of intercellular contacts and in the growth of neurites upon nerve growth factor exposure [17] PC12 cells Aminopropyltriethoxysilane-coated Fe3O4 NPs (100 μg/mL) Decrease in cell viability [164] PC12 cells IONs (40 μg/mL) Slight decrease in cell viability [165] PC12 cells DMSA-coated Fe 2 O 3 NPs (1.5 mM) Decrease in cell viability, decreased ability to respond to nerve growth factor and to generate neuritis, reduction in the formation of actin microfilaments [39] PC12 cells Fe 3 O 4 NPs (50 µg/mL) Decrease in cell viability [23] PC12 cells Fe 3 O 4 NPs (0.5 mg/mL) No evidence of cytotoxicity [92] A172 cells Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Decrease in cell viability [60] BE-2-C brain cells Carboxyethylsilanetriol-coated SPIONs (4 mg/mL) Decrease in cell viability [60] Rat neural progenitor cells Feridex® (100 µg/mL) No evidence of cytotoxicity [166] Rat brain Fe 3 O 4 NPs (500 µg/mL) No alteration of mitochondrial respiratory chain complexes I, II, III and IV activities [139] Rat brain Combidex® (10 mg/kg) (low molecular weight dextran-coated USPION), Ferumoxytol (polyglucose sorbitol carboxymethylester-coated USPIONs) (10 mg/kg) and Feridex IV® (10 mg/kg) No evidence of cytotoxicity [167] Rat brain Fe 2 O 3 NPs (1000 mg/kg) Inhibition of acetylcholinesterase [49] Mouse hippocampus α-Fe 2 O 3 NPs (130 μg per day for 30 days) Decrease in the contents of monoamine neurotransmitter and related metabolites [168] I. General Introduction 84 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Mouse brain α-Fe 2 O 3 NPs (6.5 g/L per day for 40 days) and γ-Fe2O3 NPs (6.5 g/L per day for 40 days) Morphological changes: irregular arrangement of neuron cells or neuronal loss in olfactory bulb, decrease in number of intact neuronal cells in the region I of hippocampus proper, cellular swelling, vacuolar degeneration, nuclear chromatin condensation and fragmentation [162] Murine microglial cells Resovist® (50 µg/mL) No evidence of cytotoxicity [169] Rat astrocytes Dextran-coated Fe 3 O 4 NPs (500 μg/mL) No evidence of cytotoxicity [170] Rat astrocytes DMSA-coated IONs (1 mM) Slight decrease in cell viability [171] Mouse primary astrocytes Aminosilane-coated Fe 3 O 4 NPs (224 μg/mL) Decrease in cell viability [105] Mouse primary astrocytes Carboxyaminosilane-coated Fe 3 O 4 NPs (100 μg/mL) Decrease in cell viability [105] Mouse primary neurons Aminosilane-coated Fe 3 O 4 NPs (224 μg/mL) Decrease in cell viability [105] Mouse primary neurons Carboxyaminosilane-coated Fe 3 O 4 NPs (150 μg/mL) Decrease in cell viability [105] Mouse olfactory bulb α-Fe 2 O 3 NPs (130 μg per day for 30 days) Slightly dilated rough endoplasmic reticulum, degeneration of neurodendron, disruption of membranous structure, and increase of lysosome of nerve cells [168] Lymphocytes Human peripheral lymphocytes Oleate-coated Fe 3 O 4 NPs (75 μg/cm2) Decrease in cell viability [61] A3 human T lymphocytes Amine-coated IONs (25 μg/mL) and carboxyl-coated IONs (25 μg/mL) Decrease in cell viability [52] Jurkat cells Poly(maleic anhydride-alt-1octadecene and methoxy-PEG-coated Fe3O4 NPs (150 μg/mL) Decrease in cell viability [54] Jurkat cells Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Decrease in cell viability [60] TK6 lymphoblastoid cells Oleate-coated Fe 3 O 4 NPs (45 μg/cm2) Decrease in cell viability [61] Cervical cancer cells Human cervical cancer HeLa cells Polymeric liposomes-coated Fe 3 O 4 NPs (1200 μg/mL) No evidence of cytotoxicity [172] Human cervical cancer HeLa cells PLL-coated Ferumoxides (50 μg/mL) No evidence of cytotoxicity [173] Human cervical cancer HeLa cells Fe 3 O 4 NPs (10 μg/mL) Decrease in cell viability [75] I. General Introduction 85 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Human cervical cancer HeLa cells Protamine-sulfate Feridex IV® (50 μg/mL) No evidence of cytotoxicity [141] Human cervical cancer HeLa cells Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Decrease in cell viability [60] Human cervical cancer HeLa cells Dextran-coated Fe 3 O 4 NPs (500 μg/mL), aminodextran-coated Fe3O4 NPs (500 μg/mL) and DMSA-coated Fe3O4 NPs (500 μg/mL) No evidence of cytotoxicity [174] Human cervical cancer HeLa cells Fe 3 O 4 NPs (50 µg/mL), arginine and chitosan-coated Fe3O4 NPs (50 µg/mL) and arginine and PEG-coated Fe3O4 NPs (50 µg/mL) No evidence of cytotoxicity [175] Human cervical cancer HeLa cells Carbon-coated IONs (100 μg/mL) Decrease in cell viability [131] Adipose cells Human adipose derived stem cells Protamine sulphate, PEG and dextrancoated γ-Fe2O3 NPs (15 mM) No evidence of cytotoxicity [176] Rat adipose derived stem cells Protamine sulphate-coated Ferumoxides (100 μg/mL) No evidence of cytotoxicity [177] Pancreatic cells Beta‐TC‐6 cells PVP‐coated SPIONs and Feridex® (200 µg/mL) No evidence of cytotoxicity [178] Panc-1 cells Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Slight decrease in cell viability [60] Capan-2 cells Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Slight decrease in cell viability [60] Murine insulinoma βTC3 Citrate-coated γ-Fe 2 O 3 NPs (20 µg/mL) No evidence of cytotoxicity [179] βTC-tet cells Citrate-coated γ-Fe 2 O 3 NPs (20 µg/mL) No evidence of cytotoxicity [179] Spleen Mouse splenocytes Resovist® (100 µg/mL) No evidence of cytotoxicity [180] Ovarian cells Ovarian carcinoma (MLS) cells USPIONs, arginine-glycine-aspartic acid-coated USPIONs (0.03 μmol/mL) No evidence of cytotoxicity [156] Chinese Hamster Ovary (CHO-K1) cells SPIONs (80 µg/mL) Decrease in cell viability [181] Liver cells Human hepatocytes Endorem® (150 µg/mL) Decrease in cell viability and in albumin production [182] Human primary hepatocytes Chitosan and linoleic acid-coated Fe3O4 NPs (100 µg/mL) Slight decrease in cell viability [183] HepG2 hepatocytes High density lipoprotein-coated FeO NPs (4 μg/mL) No evidence of cytotoxicity [130] I. General Introduction 86 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects HepG2 hepatocytes Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Decrease in cell viability [60] Normal mouse liver NCTC 1469 cell line Resovist® (0.5 mM) Decrease in cell viability [184] Rat liver derived BRL 3A cell line Fe 3 O 4 NPs (250 µg/mL) Decrease in cell viability [185] Hepatic cell line LO2 O-carboxymethyl chitosan-coated Fe3O4 NPs (1000 µg/mL), folic acidcarboxymethyl chitosan-coated Fe3O4 NPs (1000 µg/mL) and dextran-coated Fe3O4 NPs (1000 µg/mL) No evidence of cytotoxicity [66] Hepatic cell line LO2 Fe 3 O 4 NPs (200 µg/mL) Decrease in cell viability [66] Mouse liver Fe 3 O 4 NPs (10 mg/kg per day for 1 week) Cell expansion, liver cords broadening, liver sinuses contraction and hepatic damage [186] Murine hepatocytes Fe 2 O 3 NPs (250 µg/mL) Decrease in cell viability [187] Rat liver Fe 3 O 4 NPs (500 µg/mL) No alteration of mitochondrial respiratory chain complexes I, II, III and IV activities [139] Rat liver Fe 2 O 3 NPs (500 mg/kg) Increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels [49] Rat liver GEH121333 (200 mg/kg) No evidence of cytotoxicity [140] Rat liver Sodium oleate-coated Fe 3 O 4 NPs (0.0364 mg/kg) Diffuse necrosis and mild lipidosis [188] Fibroblasts Human fibroblasts DMSA-coated γ-Fe 2 O 3 NPs (10 µg/L) Decrease in cell viability and in metabolic mitochondrial activity [189] Human fibroblasts MION-47 and TCL SPIONs (1 mg/mL) No evidence of cytotoxicity [102] Human fibroblasts PAA and bacitracin-coated Fe 3 O 4 NPs (400 µg/mL) No evidence of cytotoxicity [190] Human skin fibroblast Poly(γ–glutamic acid)-coated Fe 3 O 4 NPs (1 mg/mL) No evidence of cytotoxicity [191] Human diploid fibroblast Gelatin-coated Fe 3 O 4 NPs (600 µg/mL) No evidence of cytotoxicity [192] Human diploid fibroblast Fe 3 O 4 NPs (300 µg/mL) Decrease in cell viability [192] Mouse fibroblast adhesive cells (L929) Fe 3 O 4 NPs (20 mM) Decrease in cell viability [193] Mouse fibroblast adhesive cells (L929) PVA-coated Fe 3 O 4 NPs (80 mM) No evidence of cytotoxicity [193] Mouse fibroblast adhesive cells (L929) Fe 3 O 4 NPs (100 mM) Decrease in cell viability [194] Mouse fibroblast adhesive cells (L929) PVA-coated Fe 3 O 4 NPs (400 mM) No evidence of cytotoxicity [194] Mouse fibroblast adhesive cells (L929) Alginate-coated Fe 3 O 4 NPs (100 µg/mL) No evidence of cytotoxicity [122] I. General Introduction 87 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Mouse fibroblast adhesive cells (L929) Poly(L-lactide)-PEG-poly(L-lactide)- coated Fe3O4 NPs (1 mg/mL) and Fe3O4 NPs (2 mg/mL) No evidence of cytotoxicity [195] Mouse fibroblast adhesive cells (L929) Dextran-coated SPIONs (1 mg/mL) Decrease in cell viability [196] Mouse fibroblast adhesive cells (L929) Surfactin-coated Fe 3 O 4 NPs (400 μg/mL) and rhamnolipid-coated Fe3O4 NPs (400 μg/mL) Decrease in cell viability [197] Mouse fibroblast adhesive cells (L929) PEG-coated Fe 3 O 4 NPs (400 μg/mL) and dextran-coated Fe3O4 NPs (400 μg/mL) No evidence of cytotoxicity [197] Mouse fibroblast adhesive cells (L929) Silica-coated γ-Fe 2 O 3 NPs (200 µg/mL) Slight decrease in cell viability [9] Telomerase immortalised primary human fibroblasts (h-TERT BJ1) Fe 3 O 4 NPs (50 µg/mL) Decrease in cell viability [46] Telomerase immortalised primary human fibroblasts (h-TERT BJ1) Pullulan-coated Fe 3 O 4 NPs (2 mg/mL) No evidence of cytotoxicity [46] Telomerase immortalised primary human fibroblasts (h-TERT BJ1) Lactoferrin and sodium oleate-coated Fe3O4 NPs (1 mg/mL) and ceruloplasmin and sodium oleatecoated Fe3O4 NPs (1 mg/mL) No evidence of cytotoxicity [198] Telomerase immortalised primary human fibroblasts (h-TERT BJ1) Fe 3 O 4 NPs (250 µg/mL) Decrease in cell viability [198] Telomerase immortalised primary human fibroblasts (h-TERT BJ1) Fe 3 O 4 NPs (50 µg/mL) and dextrancoated Fe3O4 NPs (50 µg/mL) Decrease in cell viability and cell morphology/cytoskeletal organisation and cell motility changes [199] Telomerase immortalised primary human fibroblasts (h-TERT BJ1) Fe 3 O 4 NPs (50 µg/mL) Appearance of vacuoles in the cell body and aberrations on the cell membrane [200] 3T3 cell mouse embryo fibroblast cells Oleate-coated γ-Fe 2 O 3 NPs (0.7 µg/mL) No evidence of cytotoxicity [201] 3T3 cell mouse embryo fibroblast cells Poly(glycidyl methacrylate-co-PEG methyl ether methacrylate-coated Fe3O4 NPs (1 mg/mL) No evidence of cytotoxicity [119] 3T3 cell mouse embryo fibroblast cells Silica-coated Fe 3 O 4 NPs (1 mg/mL) No evidence of cytotoxicity [6] 3T3 cell mouse embryo fibroblast cells Oleic acid-coated Fe 3 O 4 NPs (19.7 μg/cm2) Decrease in cell viability [202] 3T3 cell mouse embryo fibroblast cells Fe 3 O 4 NPs (316 μg/mL) and Endorem® (316 μg/cm2) No evidence of cytotoxicity [202] I. General Introduction 88 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects 3T3 cell mouse embryo fibroblast cells Fe 3 O 4 NPs (50 µg/mL), arginine and chitosan-coated Fe3O4 NPs (50 µg/mL) and arginine and PEG-coated Fe3O4 NPs (50 µg/mL) No evidence of cytotoxicity [175] Embryo fibroblast cells (NIH/3T3) Fe 3 O 4 NPs (50 µg/mL) Decrease in cell viability [203] Embryo fibroblast cells (NIH/3T3) PVP-coated Fe 2 O 3 /Fe 3 O 4 NPs (10 mM) No evidence of cytotoxicity [204] Embryo fibroblast cells (NIH/3T3) Fe 2 O 3 NPs (100 µg/mL) Decrease in cell viability, focal adhesion and regulation of actin cytoskeleton, lipid biosynthetic process and the cellular lipid metabolic process alterations [205] Embryo fibroblast cells (NIH/3T3) Carbon-coated IONs (100 μg/mL) Decrease in cell viability [131] Human lung fibroblast cell line MRC-5 α-Fe 2 O 3 NPs (6.25 µg/mL) Decrease in cell viability [27] Nasopharingeal cells Human nasopharyngeal epidermoid carcinoma cells (KB cells) Citrate-coated Fe 3 O 4 NPs (2 mg/mL) No evidence of cytotoxicity [206] Human nasopharyngeal epidermoid carcinoma cells (KB cells) Acetic anhydride and 3aminopropyltrimethoxysilane-coated Fe3O4 NPs (100 µg/mL) No evidence of cytotoxicity [64] Human nasopharyngeal epidermoid carcinoma cells (KB cells) Succinic anhydride and 3aminopropyltrimethoxysilane-coated Fe3O4 NPs (10 µg/mL) and 3aminopropyltrimethoxysilane-coated Fe3O4 NPs (10 µg/mL) Decrease in cell viability [64] Human nasopharyngeal epidermoid carcinoma cells (KB cells) Pluronic F127 and PAA-coated Fe 3 O 4 NPs (1 mg/mL) No evidence of cytotoxicity [207] Human nasopharyngeal epidermoid carcinoma cells (KB cells) Oleic acid and PEG-coated Fe 3 O 4 NPs (200 µg/mL) No evidence of cytotoxicity [208] Human nasopharyngeal epidermoid carcinoma cells (KB cells) Fe 3 O 4 NPs (100 µg/mL) Decrease in cell viability [66] Human nasopharyngeal epidermoid carcinoma cells (KB cells) O-carboxymethyl chitosan-coated Fe3O4 NPs (1 mg/mL) and dextrancoated Fe3O4 NPs (1 mg/mL) No evidence of cytotoxicity [66] Erythrocytes Rabbit erythrocytes Alginate-coated Fe 3 O 4 NPs (570 µg/mL) No evidence of cytotoxicity [122] Rat erythrocytes Fe 2 O 3 NPs (500 mg/kg) Inhibition of acetylcholinesterase [49] Human erythrocytes Tetramethylammonium hydroxidecoated Fe3O4 NPs (0.1 M) Increase in hemolysis [209] Human erythrocytes Acetic anhydride and 3aminopropyltrimethoxysilane-coated Fe3O4 NPs (400 µg/mL) No evidence of cytotoxicity [64] I. General Introduction 89 Table 1. Toxic effects of IONs in several cell types and tissues (continuation). Type of cell/tissue Type and concentrations of nanoparticles Cytotoxic effects Human erythrocytes Succinic anhydride and 3aminopropyltrimethoxysilane-coated Fe3O4 NPs (400 µg/mL) and 3aminopropyltrimethoxysilane-coated Fe3O4 NPs (400 µg/mL) Increase in hemolysis [64] Human erythrocytes Poly(L-lactide)-PEG-poly(L-lactide)- coated Fe3O4 NPs (20 mg/mL) and Fe3O4 NPs (20 mg/mL) Increase in hemolysis [195] Human erythrocytes Folic acid-PEG-PEI-coated Fe 3 O 4 NPs (400 μg/mL) No evidence of cytotoxicity [120] Kidney cells Human embryonic kidney 293T cell line Carboxyethylsilanetriol-coated SPIONs (8 mg/mL) Decrease in cell viability [60] Human embryonic kidney 293T cell line PEI-PAA-coated Fe 3 O 4 NPs (200 µg/mL) No evidence of cytotoxicity [210] Cos-7 monkey kidney cells Tetramethylammonium hydroxidecoated Fe3O4 NPs (23.05 mM) No evidence of cytotoxicity [209] MDCK (canine distal tubule) epithelial cell line Oleic acid-coated Fe 3 O 4 NPs (235 µg/mL) Decrease in cell viability [211] LLC-PK (porcine proximal tubule) epithelial cell line Oleic acid-coated Fe 3 O 4 NPs (235 µg/mL) Decrease in cell viability [211] Human urinary bladder carcinoma cells UM-UC-3 Poly(ε-caprolactone)-b-poly(propargyl methacrylate-co-poly- (ethylene glycol) methyl ether methacrylate)-coated SPIONs (1 mg/mL) No evidence of cytotoxicity [212] Renal NRK cells Dextran-coated Fe 3 O 4 NPs (500 μg/mL) No evidence of cytotoxicity [170] Mouse kidney Fe 3 O 4 NPs (5 mg/kg per day for 1 week) Large reduction of tubular space and extreme edema of epithelial cells in glomeruli [186] Mouse kidney PAA-coated γ-Fe 2 O 3 NPs (10 mg/kg) No evidence of cytotoxicity [213] Rat kidney Fe 3 O 4 NPs (500 µg/mL) No alteration of mitochondrial respiratory chain complexes I, II, III and IV activities [139] Rat kidney Fe 2 O 3 NPs (500 mg/kg) Decrease in AST and ALT levels [49] Rat kidney Fe 2 O 3 NPs (1000 mg/kg) Decrease in LDH levels [49] Rat kidney GEH121333 (200 mg/kg) No evidence of cytotoxicity [140] Muscular cells C2C12 cells Protamine sulfate-coated Feridex® (30 μg/mL) No evidence of cytotoxicity [214] Embryonic cells Embryonic stem cells PLL-coated SPIONs (50 μg/mL) No evidence of cytotoxicity [215] I. General Introduction 90 cell line to the elevated ROS production. It is known that ROS interact with cellular biomolecules such as proteins, membrane lipids and even DNA which could be oxidized, destructured and ultimately became non-functional [33]. In fact, it is thought that the enhanced production of ROS caused by mitochondria or membrane-bound NADPH oxidases in response to their interaction with IONs might be a possible mechanism involved, causing damage to both purine and pyrimidine bases as well as the DNA backbone [32,240]. Moreover, iron ions released from IONs participate in Fenton reaction, originating HO•, which is highly reactive, leading to an increase in DNA damage, attacking a multitude of oxidized bases, namely DNA base guanine to form 8-hydroxy-2′- deoxyguanosine adducts, which are known to have mutagenic potential, abasic sites, single and double-strand breaks [33,77,239]. An example of this has been described by Ma et al [186]: Fe3O4 NPs increased 8-hydroxy-2′-deoxyguanosine adducts in mouse hepatic and renal tissues. RNS are also able to induce DNA damage: •NO can react with O2•-, producing ONOO-, which in turn can generate other RNS to interact with proteins and nucleic acids [241]. The coating and the extent of uptake are described as exerting a determinant role concerning genotoxicity. Singh et al [239] stated that cellular internalization of IONs is positively correlated with the induction of genotoxicity. This author also showed that, while dextran-coated γ-Fe2O3 NPs induced DNA damage in human B-lymphoblastoid MCL5 cells, γ-Fe2O3 NPs, dextran-coated Fe3O4 NPs and Fe3O4 NPs did not exert the same effect [239]. Similarly, Hong et al [242] reported that, in mouse fibroblast adhesive cells (L929), (3-aminopropyl)trimethoxysilane-coated Fe3O4 NPs, tetraethyl orthosilicate-(3aminopropyl)trimethoxysilane-coated Fe3O4 NPs and citrate-coated Fe3O4 NPs triggered DNA damage, while non-coated Fe3O4 NPs and tetraethyl orthosilicate-coated Fe3O4 NPs did not exert such effect. The fact that (3-aminopropyl)trimethoxysilane-coated Fe3O4 NPs and tetraethyl orthosilicate-(3-aminopropyl)trimethoxysilane-coated Fe3O4 NPs induce genotoxicity is probably due to the fact that these positively charged IONs are more concentrated within the cells and enter into the nucleus through the nuclear pore and interact directly with the DNA, which is negatively charged due to its phosphate groups [242]. Citrate-coated Fe3O4 NPs are shown to penetrate through nuclear membrane, generating highly reactive HO• in close proximity to DNA, leading to DNA attack [242]. Chen et al [195] reported that both poly(L-lactide)–PEG–poly(L-lactide)-coated Fe3O4 NPs and Fe3O4 NPs increased micronuclei in Chinese Hamster Ovary (CHO-K1) cells, although this increase has been more pronounced in the case of Fe3O4 NPs. The authors defend that this polymer coating may reduce the DNA damage caused by the IONs, I. General Introduction 97 affecting the physical presence and/or disturbance of the NPs around the mitotic apparatus [195]. Likewise, Magdolenova et al [61] found that oleate-coated Fe3O4 NPs induced DNA damage in human peripheral lymphocytes and TK6 lymphoblastoid cells but Fe3O4 NPs did not exerted the same effect. Nevertheless, the authors reported that oleate-coated Fe3O4 NPs exhibited lower cellular uptake than Fe3O4 NPs. This fact proves that augmented IONs internalization is not always synonym of increased genotoxicity. Coating of Fe3O4 NPs with oleate was found to change the genotoxic potential of the Fe3O4 NPs, probably by inducing changes in features of the IONs related to cellular uptake and binding to extracellular receptors or transmembrane channels [61]. Noncoated IONs have also demonstrated to be cytotoxic: Fe2O3 NPs induced DNA damage in human lung fibroblasts IMR-90 cells and human lung epithelial cells BEAS-2B [77,145]. Fe3O4 NPs induced DNA damage in human lung cancer A549 cells and human epithelial cell line A-431 [33,68]. In effect, the cells involved and the type of interactions between the cells and ION may be determinant for the genotoxicity. Besides that, it is also known that genotoxicity may suffer interindividual variations. Novotna et al [65] reported that, although PLL-coated γ-Fe2O3 NPs, PLL-coated Endorem®, mannose-coated γ-Fe2O3 NPs, poly(N,N-dimethylacrylamide)-coated γ-Fe2O3 NPs and γ-Fe2O3 NPs have induced DNA damage in human bone marrow mesenchymal stromal cells of the two individuals studied, Endorem® only induced DNA damage in one of the individuals, demonstrating not to be genotoxic in the other individual tested. I.1.2.4. PRO-INFLAMMATORY EFFECTS OF IRON OXIDE NANOPARTICLES Many studies have been performed in the last few years to evaluate the impact and/or the effect of IONs on the different cells and animal models regarding inflammation [180,243,244]. The interaction between NPs and immune cells and the consequences of such interactions are relevant issues in addressing the potential impact of IONs on inflammatory processes [245]. It is important to remember that the cellular recruitment processes are mediated by a battery of proinflammatory cytokines, including interleukin (IL)-8, IL-6, tumor necrosis factor α (TNF-α), etc. that induce increased microvascular caliber, enhance vascular permeability, leukocyte recruitment, and release of inflammatory mediators (figure 1). This process is of relatively short duration, lasting from several minutes to a few days. However, when inflammation becomes chronic, it can result in tissue destruction and lead to organ dysfunction in pathological situations I. General Introduction 98 [246,247]. IONs have been described as being involved in the different stages of the inflammatory process. Therefore, in this chapter, the effect of IONs on the most relevant inflammatory pathways, as well as in the cytokine production, is reviewed. I.1.2.4.1. MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) PATHWAY MAPK signaling pathways relay, amplify and integrate signals from a diverse range of extracellular stimuli, thereby controlling the genomic and physiological response of a cell to changes in the environment. It is a critical link between cell surface signal transduction and nuclear processes, including cell proliferation, differentiation and migration, development, inflammatory response and apoptosis [248]. Mammalian cells express at least four distinctly regulated groups of MAPK, specifically extracellular signal-related kinases (ERK)-1/2, JNK1/2/3, p38 proteins (p38 α/β/γ/δ) and ERK5, which are activated by different MAPK kinases (MKK) [249]. Liu et al [90] reported that DMSA-coated Fe3O4 NPs activate several members of the MAPK family in RAW264.7 cells. DMSA-coated Fe2O3 NPs, however, decreased MAPK14 (or p38α) expression in human aortic endothelial cells [98], showing the differential responses that may occur when different cells as well as different type of IONs are studied. ERK1/2 are distributed throughout quiescent cells, but upon stimulation, a significant population of ERK1/2 accumulates in the nucleus. In fact, upon receptor activation, membrane-bound Ras recruits one of the Raf kinases into a complex where it becomes activated. Afterwards, Raf phosphorylates two serine residues on the kinase mitogen protein kinase kinase (MEK) 1 and 2, which in turn activate ERK1/2 by tandem phosphorylation of threonine and tyrosine residues on the dual-specificity motif. While the mechanisms involved in nuclear accumulation of ERK1/2 remain elusive, nuclear retention, dimerization, phosphorylation, and release from cytoplasmic anchors have been shown to play a role. Activated ERK1 and ERK2 phosphorylate numerous substrates in all cellular compartments, including several MKK [250,251]. Moreover, stimulation of inflammatory cells and ROS also triggers protein phosphorylation, where MKK are activated via phosphorylation by MKK kinases (MAPKKK), and these MKK, usually MKK 3 or 6, lead to phosphorylation of p38 MAPK and JNK [252]. JNK and p38 represent one I. General Introduction 99 subgroup of MAPK that is activated primarily by cytokines and exposure to environmental stress. A major target of the these signaling pathways is the activation of the activator protein 1 (AP-1) transcription factor that is mediated, in part, by the phosphorylation of cJun and related molecules, such as activating transcription factor 2 (ATF-2) and c-Fos, resulting in the production and secretion of pro-inflammatory cytokines, such as IL-1β and TNF-α [248,252]. AP-1 levels were shown to be increased by dextran-coated Fe3O4 NPs in the JB6 P+ murine epidermal cell line [253]. Therefore, in this case and in the most of the cases above mentioned, a pro-inflammatory response is expected to be increased. It is also described that Resovist® significantly induced ERK1/2 and Akt phosphorilation in pancreatic β cells [254]. In fact, ERK1/2 are components of the mechanism by which glucose stimulates insulin gene expression, which is mediated by the activation of transcription factors (pancreatic and duodenal homeobox 1, neurogenic differentiation 1 and E47) [255]. It was also described that PEG-coated Fe3O4 NPs increased the phosphorylation of ERK1/2 in PC12 cells in the presence of nerve growth factor [165] and γ-Fe2O3 NPs and Fe3O4 NPs increased p-ERK levels in RAW 264.7 cells [48]. ERK1 is also essential for regulation of energy homeostasis together with p62 [256] and, for this reason Park et al [48] suggest that the increase in p-ERK triggered by γ-Fe2O3 NPs was activated for cell survival. Moreover, Resovist® and Supravist®-induced JNK activation in human macrophages was associated with an increase in apoptosis, given that it is known that JNK may lead to formation of the proapoptotic Bid protein [132]. Fe3O4 NPs was also shown to activate JNK in a ROS-independent manner in A549 cells [32], although Park et al [48] reported that γ-Fe2O3 NPs and Fe3O4 NPs decrease the levels of p-JNK in RAW 264.7 cells. I.1.2.4.2. NUCLEAR FACTOR kB (NF-kB) SIGNALING PATHWAY NF-kB is a pivotal mediator, which is involved in multiple cellular responses. This transcription factor could be activated by a variety of stimuli, including cytokines and ROS, and regulates the transcription of various proand inflammatory mediators, namely cytokines (IL-1β, TNF-α, and IL-6), chemokines [IL-8 and macrophage inflammatory protein 2 (MIP-2)], adhesion molecules [vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin], and enzymes [inducible nitric oxide synthase (iNOS)] [32,257,258]. In unstimulated cells, NF-kB is retained in the cytosol by inhibitor of kB (IkB). Stimulation with TNF-α or foreign agents leads to I. General Introduction 100 degradation of IkB which then dissociates from NF-kB enabling it to translocate to the nucleus where it exerts its transcriptional function, increasing the transcription of several cytokines, such as TNF-α, IL-1, IL-6 and IL-8 [32,259]. Zhou et al [150] reported a slight increase in NF-kB-DNA binding activity caused by Fe2O3 NPs in rat lung. However, Fe3O4 NPs did not trigger any alteration on NF-kB activity in rat primary microglia [92], human lung cancer A549 cells [32], occurring a decrease and delay in the IkB degradation in the latter. Therefore, in most of the cases above mentioned, NF-kB does not seem to be an inflammatory pathway largely activated by IONs, as it occurred for MAPK pathways. Consequently, cytokine production triggered by IONs seems to be due to the involvement of other pathways, namely MAPK pathways. I.1.2.4.3. CYTOKINES Cytokines are a group of soluble proteins, peptides or glycoproteins regulators of host responses to infection, immune responses, inflammation, and trauma. There are several cytokines that are produced in our organism in the events above mentioned, namely human TNF-α, interferon γ (IFN-γ), IL-1, IL-6, among others [260]. Cytokines may be produced via regulation of transcription factors, namely NF-kB and MAPK pathways as well as due to monocyte/macrophage activation in response to microbial products, which gives rise to the activation of T-cell immunity. Classical activation of macrophages, defined as macrophages subset 1 (M1) activation, may promote the differentiation of Thelper lymphocytes 1 (Th1) cells, whereas macrophages subset 2 (M2) activation could promote T-helper lymphocytes 2 (Th2)-type responses. In both types of responses, there is cytokine production. M1 activation is activated by IFN-γ and characterized by elevated phagocytic ability to kill pathogens, microorganism and tumour cells, elevated expression of major histocompatibility complex (MHC) class II, generation of ROS, and production of the Th1 cytokines, such as IL-12 and TNF-α. M2 macrophages activation down-regulates Th1 responses and stimulates Th2 responses, with production of Th2 cytokines such as IL-4 and IL-6, scavenge debris, and promote angiogenesis, tissue remodelling and repair [261,262]. TNF-α, a pro-inflammatory cytokine, is synthesized and secreted by several types of cells, but especially by macrophages. TNF-α mediates a wide range of biological responses including inflammation, infection, injury, and apoptosis and has a strong antitumor effect, markedly inhibiting tumor growth by inducing tumor cell necrosis [263,264]. I. General Introduction 101 This cytokine is also involved in IL-6 as well as IL-8 production, being this latter the most potent known chemotactic agent for neutrophils [260,265,266]. Several IONs showed to activate TNF-α, namely Fe3O4 NPs in mouse BALF [267] and Resovist® in THP-1 cell line [268]. IFN-γ is a type-II IFN secreted by several immune cells (dendritic cells, Th1, T cells, and natural killer) with anti-viral, anti-tumor, and immunoregulatory effects that can be exerted at several levels. IFN-γ regulates class I and II antigen presentation through the expression of key genes related to MHC class I and II-dependent antigen-presentation [269]. IONs showed to increase this cytokine, namely Fe3O4 NPs in mouse peripheral blood [244] and Fe2O3 NPs in mouse BALF [270]. IL-1 activates a cascade of cytokine production and induces the production of a broad range of immunomodulatory cytokines. IL-6 is among the mediators regulated by IL-1 and is frequently increased in inflammatory processes [260,271]. In fact, some IONs have shown to activate IL-1β, as it is the case of DMSA-coated Fe2O3 NPs in rat sciatic nerve [223], Fe3O4 NPs in mouse BALF [267] and citrate-coated γ-Fe2O3 NPs in human gingival fibroblasts [272] and also IL-6, as it is the case of dextran-coated Fe3O4 NPs in human epidermal keratinocytes [253] and Fe2O3 NPs in mouse BALF [270], which demonstrates IONs involvement in pro-inflammatory responses. However, IL-1β may be involved in other mechanisms. In effect, IL-1β released by microglia induces the proliferation of astrocytes, stimulates neovascularization and promotes repair of the central nervous system in brain injury. Wu et al [169] showed that Resovist® attenuated IL-1β production in murine microglial cells by suppressing the secretory lysosomal functionality in LPSstimulated microglia through cathepsin B activity attenuation, which may indicate that IONs promote brain injury. Transforming growth factor beta (TGF-β) is a large family of cytokines that includes activins, inhibins, bone morphogenetic proteins and nodal and growth differentiation factors. These ligands of the TGF-β signaling pathways bind to a diversity of cell-surface receptors and cause signal transduction through the SMADs [273,274]. TGF-β1 is the most important mediator that influences collagen synthesis and other matrix molecules, activating the gene transcription of collagens I, III, IV, VI, and VII, proteoglycans and matrix metalloproteinases [272,275]. TGF-β activation has been triggered by Fe3O4 NPs in mouse BALF [267]. I. General Introduction 102 IL-10, as well as IL-4, is a cytokine considered to have an anti-inflammatory role. IL10 antagonizes a subset of genes activated by Toll-like receptors signaling, such as TNFα, IL-6, and numerous chemokine mRNA [276]. In fact, some IONs were shown to induce IL-10, inhibiting inflammatory cytokines such IL-1β, IL-6 and IFN-γ. Blank et al [277] reported that PVA-coated SPIONs triggered a decrease in IL-1β, IL-6, TNF-α and IFN-γ and increase in IL-10 levels in human monocyte-derived dendritic cells in the presence of LPS and tetanus toxoid [277]. The influence of IONs in cytokine production has been described in other different cell types [95,108,125,278,279]. The studies performed so far indicate that IONs may have several effects on cytokine production, depending on the several factors. In fact, it is known that IONs can modulate cytokine profile and this modulation may be the result of distinct mechanisms of IONs uptake by the cells. Once inside the cell, the fate of the IONs in terms of subcellular location/translocation is different depending on the IONs and on the cell. Consequently, the interactions established between the IONs and the receptors and/or the cellular machinery are different, depending on many factors such as surface charge, coating, type of IONs, among other factors. All these factors may lead to immunesuppression or immune-stimulation, modifying the mode how the antigen is handled in cells [277,280]. The animal model used may also influence the results obtained, determining the regulation of cytokine expression. Siglienti et al [281] reported that, in mouse macrophages, Resovist® and SH U 555C (carboxydextran-coated USPIONs) increased IL-10 levels, while in rat macrophages, the same IONs decreased IL-10 and TNF-α levels. I.1.2.4.4. ION-INDUCED ACTIVATION OF MACROPHAGES AND T CELLS In inflammation, the major role of monocytes/macrophages is to recognize and eliminate foreign material. For this purpose, they have three major functions: antigen presentation, phagocytosis, and immunomodulation through production of various cytokines and growth factors [89,247]. It is known that NPs in biological fluids and tissues, as other foreign agents, are frequently covered with biological molecules, namely proteins that can facilitate their interaction with monocytes/macrophages, as well as their degradation and clearance from the bloodstream [282,283]. For example, Valois et al [282] clearly demonstrated an uptake of DMSA-coated Fe2O3 NPs by I. General Introduction 103 monocyte/macrophage cells, indicating that this may be a mechanism of NP clearance used by the lung in order to avoid further damage. It is described that IONs have the potential to modify monocytes’ ability to respond to other activating stimuli, as well as to alter monocytes’ activity, namely in the recruitment, adhesion and migration into the subendothelial layer of the intima, representing a considerable risk factor for promoting early events in the development of atherosclerosis [99]. T lymphocytes include three major functional subsets: T-helper lymphocytes, Tsuppresser lymphocytes, and T-cytotoxic lymphocytes [243]. The immune response depends on the balance of cytokines produced by two Th cell subsets, Th1 and Th2. Th1 cells secrete Th1 cytokines such as IL-2 and INF-γ, promote cytotoxic and inflammatory functions by activating macrophages, natural killer cells and cytotoxic CD8+ T lymphocytes. Th2 cells secrete Th2 cytokines such as IL-4, IL-6 and IL-10, which can trigger the humoral immune mechanism, including antibody production and eosinophil proliferation [284]. There are several studies concerning the role of IONs on T cell activation. In fact, it has been described that magnetic IONs have the ability to generate a significant number of exosomes in the alveolar region of mice. In turn, the immune activation in splenic T cells is simultaneously induced [285]. Fe3O4 NPs were shown to increase CD8+ T cells activation and decrease CD4+ T cells activation [267]. When mixed lymphocyte reaction was evaluated, Endorem® revealed an inability to interfere with the dendritic cells activation of the CD8+ T cells [110], CD4+ T cells [286] and protamine sulphate-coated Feridex IV® showed an inability to interfere with the THP-1 cells activation of the CD4+ T cells [287]. However, Pawelczyk et al [288] observed, performing the same experiment, suppression of bone marrow stromal cells activation of alloreactive T-cells by protamine sulphate-coated Feridex IV®. Here, the different type of cell may have contributed for the different results. It was described, by Zhu et al [285], that, in ovalbumin-sensitized rats, magnetic IONs have the ability to increase the percentages of Th1 and T-cytotoxic lymphocytes 1, decreasing at the same time the percentages of Th2 and T-cytotoxic lymphocytes 2, which suggest that magnetic IONs are able to skew the immune response toward Th1 responses. Other authors reported that Fe3O4 NPs increase Th1 immunity in a greater extent than they increase Th2 immunity [244]. In accordance, Ban et al [289] reported that Fe2O3 NPs inhibited the ovalbumin-induced Th2 response in mice. Zhu et al [261] I. General Introduction 104 reported that IONs-induced exosomes triggered classical peritoneal macrophages activation to M1. Laskar et al [278] also reported that Resovist® induces a phenotypic shift of THP1 M2 macrophages towards M1 subtype. 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