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2014 43 Lamiaa Mohamed Ahmed Ali Toxicity Studies of Polymer Based Supermagnetic Iron Oxide Nanoparticles Departamento Director/es Física de la Materia Condensada Palacio Parada, Fernando Gutiérrez Martín, Martín Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Lamiaa Mohamed Ahmed Ali TOXICITY STUDIES OF POLYMER BASED SUPERMAGNETIC IRON OXIDE NANOPARTICLES Director/es Física de la Materia Condensada Palacio Parada, Fernando Gutiérrez Martín, Martín Tesis Doctoral Autor 2014 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Toxicity Studies of PolymerBased Superparamagnetic Iron Oxide Nanoparticles Colección de Estudios de Física Vol. XX Esta colección recoge las tesis presentadas en el Departamento de Física de la Materia Condensada de la Universidad de Zaragoza desde su constitución en 1987. Colección de Estudios de Física Vol. XX Toxicity Studies of Polymer Based Superparamagnetic Iron Oxide Nanoparticles Lamiaa Mohamed Ahmed Ali Aquí va la hoja de créditos... Thesis realised under the supervision of Prof. Fernando Palacio Parada Instituto of Ciencias de Materiales de Aragón y Departamento de Física de la Materia Condensada, Facultad de Ciencias. Prof. Martín Gutiérrez Martín Departamento de Medicina, Facultad de Medicina. XIII Preface turn, shed the light on the importance of this kind of studies, especially for nanomaterials that are developed for intravenous administration. A brief illustration for the blood coagulation mechanism, regulatory mechanisms and coagulation screening tests are also provided. The main purpose of this chapter is to study the toxicity of three different types of polymer coated SPIONs and their separated component in blood. This chapter displays the influence of the surface coating and surface charge on the toxicity behaviour of the bioferrofluids. The three types of bioferrofluids exhibit anticoagulant effect, with no effect on the blood count in vitro. No incidence of haemolysis was detected. Chapter 5 has the title of “Biodistribution studies of polymer coated superparamagnetic iron oxide nanoparticles”. This chapter starts with a brief review about the biomedical imaging and MRI, with an illustration of the basic principles of MRI and contrast agents. In addition, this chapter discusses the factors affecting the biodistribution of the nanomaterials. The aim of this chapter is to study the in vitro relaxation, in vivo biodistribution, and in vivo toxicity for two polymer coated SPIONs. The obtained results showed that our bioferrofluids are a good T2 contrast agents with no toxic effect in vivo. Chapter 1 General Introduction and Purpose of the Thesis 1.1 Nanotechnology, nanomedicine and nanotoxicology terms Nanotechnology has garnered a great interest in last decades. According to the United States (US) Nanotechnology Initiative, Nanotechnology can be defined as “the understanding and control of matter at dimensions of roughly 1–100 nanometers, where unique phenomena enable novel applications” [1]. Nanomaterials have a size comparable to the size of the sub-cellular organelles and proteins Fig. 1.1, hence, they could be exploited to spy or interfere with the cellular machinery giving rise to a new branch of science called “nanomedicine” [2]. Figure 1.1: Demonstration of the nanomaterials sizes compared with other items [3]. 2 Chapter 1. Introduction The European Science Foundation (ESF) in its Forward Look Nanomedicine document defined nanomedicine as the following “nanomedicine uses nano-sized tools for the diagnosis, prevention and treatment of disease and to gain increased understanding of the complex underlying patho-physiology of disease” [4]. Although, the unique physicochemical properties of nanomaterials have a great positive impact on biomedical applications, the same properties can have a negative impact on the biosystem, since nanomaterials are characterised by a high surface to volume ratio, which may lead to increase their biological reactivity. In addition, their nano-size enables them to breach cells reaching critical sites inside, such as nucleus, mitochondria, etc., which may cause mutations or cell death [5]. Besides the environmental exposure to airborne nano-sized materials coming from natural or anthropogenic sources [6], the rapid evolution in nanotechnology, which extends to many applications (biological, medical and industrial) [5,7,8], increases the risk of exposure. The issue of nanomaterials safety and nanotoxicity should be addressed, as a lesson learned from the medical science´s failure in the past centuries to take safety measures toward quartz and asbestos exposure [9]. Therefore, a new sub-discipline of the nanotechnology called nanotoxicology has emerged. Nanotoxicology could be defined as “science of engineered nanodevices and nanostructures that deals with their effects in living organisms” [6]. We can conclude that nanotechnology, nanomedicine and nanotoxicology are three interconnected and closely dependent fields. Therefore, as any new nanomaterial can have an impact on the biological environment with consequences in the medical field, a series of toxicology studies should be carried during its development process. Therefore, for any nanomaterial developed for biomedical applications, toxicological studies should include: synthesis and characterisation with the possibility to minimise batch to batch variation, studying nanomaterial toxicity on cells (in vitro), studies of nanomaterial uptake and mechanism of internalisation (endocytosis), studies of nanomaterial toxicity on blood (haemotoxicity) particularly if the nanomaterial is developed for intravenous (iv) injection, studies of the nanomaterial biodistribution (in vivo) and their possible toxic effects on tissues and organs, evaluation of their 1.2. Nanomedicine 3 application (therapeutic or diagnostic) efficiency and finally studying their toxic effect on the environment. As explained in more detail at the end of this chapter, this thesis will deal with some of these studies as a necessary step in the group for the development of a synthetic nanoplatform for biomedical applications. Along the thesis it will become clear that toxicological issues often depend of properties that are independent of the materials composition, e.g, aggregation and surface charges, in addition to their composition itself. Additionally, nanomaterials can be rather complex systems with a variety of components whose individual toxicity should also be addressed. There is therefore an interrelation between the nanomaterial, its components toxicity and its effects on the biological system it is being developed for. In the following sections we will revise each of these four fundamental aspects: nanomedicine, magnetic nanoparticles, nanomaterials and their design and nanotoxicity. 1.2 Nanomedicine: a new way to address open medical and pharmaceutical problems There are many medical problems still waiting to be solved that are associated either with the diagnosis and/or with the treatment of some diseases. For instance, in the case of cancer, early diagnosis is considered a challenge in its treatment, which in many cases is not reached. Moreover, traditional cancer treatment methods (radiotherapy or chemotherapy) are associated with several complications such as the lack of efficient selectivity toward cancerous cells causing systemic toxicity [10]. Another treatment limitation is the inability of chemotherapy to overcome the multidrug resistance (MDR) mechanism -a mechanism of drug efflux from the cancerous cell cytoplasm to the extracellular spacethat is associated with several tumour cells leading to treatment failure [11]. Nanomedicine opens new hopes in solving diagnostic and therapeutic problems by developing nanomedicines -an overall term that includes nanopharmaceuticals, 4 Chapter 1. Introduction nanoimaging agents, and theranostics1 [12]-, and exploiting new physicochemical properties, since at the nano-level the material can acquire new properties (electrical, optical or magnetic) not present in the bulk state [13]. Nanomedicines can be organic such as liposomes, micelles, dendrimers, polymeric nanoparticles, fullerenes and carbon nanotubes or inorganic such as magnetic nanoparticles, metal based nanoparticles, ceramic nanoparticles and semiconductors. This versatility in composition and physicochemical properties opens the door for a wide range of medical applications [14]. Therefore, the second half of the 20th century witnessed the birth of the first generation of marketed nanomedicines, their use is varying between diagnosis and therapy, as shown in Table 1.1 where only those related to iron oxide are collected. A rather comprehensive account of nanomedicines already in the market can be found in ref. [12]. 1.2.1 Therapeutic nanomedicines The onset of the use of nanomedicines as therapeutic agents dates back to fifties, when the first polymer-drug-conjugate was synthesised by Jatzkewitz. This was a breakthrough in the manufacturing of nanomedicines as depicted in Fig. 1.2 [15]. The use of nanomaterials as therapeutic agents is a diverse field, ranging from drug/gene delivery systems, antimicrobial agents to antioxidant agents [2,12,16,17]. Traditional delivery strategies for certain drugs (especially anticancer) are associated with many prominent problems such as lack of selectivity which in turns is reflected on the toxicity, failure to avoid MDR mechanism, low bioavailability, the necessity of large dose to achieve high local concentration and fail to improve drug solubility [18]. Nanomedicine reduces these previous problems improving drug delivery strategies. This is achieved by drug encapsulation, surface conjugation or entrapment to the nanomaterials forming “targeted” drug delivery systems. As a result, these approaches transport drugs more efficiently to the target site, increase the local concentration of the drug in the target site, improve drug solubility 1 Theranostic is a nanomedicine concept which refers to the capacity of a product to act as a diagnostic and therapeutic tool simultaneously. 1.2. Nanomedicine 5 Table 1.1: First generation of marketed iron oxide-based nanomedicines [12]. Product name Technology Indication Route of admin. Information source Superparamagnetic Iron Oxide Imaging Nanoparticles (SPIONS) Feridex (ferumoxide) Endorem (ferumoxide) Gastromark (ferumoxsil) Lumirem (ferumoxsil) Sinerem (ferumoxtran), same as Combidex Resovist SPION dextran coating SPION dextran coating SPION silicone coating SPION silicone coating ultrasmall particles (USPION) SPIONs liver imaging liver imaging GI imaging GI imaging lymph node imaging small liver lesions iv iv oral suspension oral suspension infusion iv Bayer Healthcare Pharmaceuticals AMAG Pharmaceuticals/Guerbet SAa AMAG Pharmaceuticals a AMAG Pharmaceuticals a Guerbet withdrew MAA for Sinerem in 2007 AMAG Pharmaceuticals (phase III) Bayer Healthcare Pharmaceuticals Iron Oxide Supplements Venofer Ferrlecit Cosmofer various “generic” formulations iron oxide, sucrose iron oxide gluconate iron oxide, dextran iron oxide nanoparticles anemia anemia anemia anemia iv iv iv iv Freseniusa Sanofi-Aventisa GRY-Pharmaa reviewed in refs 508, 509 6 Chapter 1. Introduction and protect the drug from degradation. They also open the possibility to deliver a combination of drugs for a combination therapy, or to monitor the drug delivery by delivering a combination of imaging and therapeutic agents, and therefore all these previously mentioned advantages lead to increase patient compliance [18,19]. Drug targeting using nanomedicines is achieved by either passive or active targeting mechanism. Passive targeting is divided into two mechanisms: a) enhanced permeability and retention (EPR) mechanism, which exploits the leaky vasculature of tumour cells [20], b) localised delivery mechanism by intra-tumour injection of drugs conjugated to nanomaterials [21]. However, active targeting is achieved by direct conjugation of the nanomaterials to various signature molecules2 recognisers such as ligand [22], antibody [23] or aptamer [24], in order to trigger nanomedicines to specific pathological site. All the first generation marketed nanomedicines are passively targeted. For instance, Doxil (liposomes-encapsulated doxorubicin), was approved by the US Food and Drug Administration (FDA) in 1995. It triggered drug targeting through EPR mechanism and was used for the treatment of advanced ovarian cancer, metastatic breast cancer and HIV-treated Kaposi´s sarcoma [15]. Although more than 30 years have passed since the first description of ligand conjugated liposomes in 1980, only a handful of active targeted nanomedicines have reached to the clinical trial area, and none have made a significant clinical impact on human health [12,25]. Continual advances in nanofabrication lead to production of nanomedicines not only able to protect the cargo from unfavourable conditions but also controlling its release as a response to physical or chemical stimuli such as light, heat, sonication, magnetic field, pH and redox change [26]. In addition, elongation for blood circulation time could be achieved through polymer coating [27]. An additional merit for nanomedicines as targeted drug delivery system is their ability to deliver drugs across blood brain barrier (BBB) [28]. The delivery property of nanomedicines is not limited to drugs only but also extended to genes [29] and 2 Signature molecules are biological molecules found in blood, other body fluids or tissues, which are signs of a normal or abnormal process, or of a condition or disease. 1.2. Nanomedicine 7 Figure 1.2: Timeline of nanomedicines development [15]. 8 Chapter 1. Introduction Figure 1.2 (continue): Timeline of nanomedicines development [15]. 1.3. Magnetic nanoparticles 15 Charité Hospital, led by Andreas Jordan, has been developing hyperthermia using MNPs since 1993 [65]. Nowadays, there are Clinical trials in phase II in Germany to perform hyperthermia on human patients using IONPs by direct injection hyperthermia (DIH) into two kinds of tumours: glioblastoma and prostate [66–69]. It has been reported that the nanoparticle physicochemical properties, such as size [70], surface coating [69] and charge [71] affect the specific absorption rate (SAR), which indicates the heat evolution rate in hyperthermia. In the case of magnetite, the SAR of 35 nm particles is much higher than that of 10 nm particles [70,72]. Till now, hyperthermia is commonly based on the direct injection of highlyconcentrated NPs solutions into solid tumours, since the tumour passive-targeting of NPs leads to quite low particles concentration in the tumour cells. Therefore, a new trend geared toward synthesising specific targeted NPs for hyperthermia has emerged [73]. The problem of the systemic administration of these compounds is the low particle concentration within the tumour. To overcome this problem is at the forefront of current research, either by developing more efficient MNPs solution or by improving targeting. 1.3.1.1.2.2 Drug delivery Conventional drug delivery is associated with several disadvantages such as low local drug concentration, systemic toxicity, etc. Therefore, development of new pharmaceutical techniques for targeted drug delivery has gained immense attention. MNPs are considered as a promising tool for targeted drug delivery. The magnetic functionality enables MNPs to be guided to certain target site using an external magnetic field gradient. Targeting can also be achieved through direct conjugation of a target moiety (antibody, peptide or aptamer) to the MNP surface. Several studies showed the ability of the MNPs to deliver the drug to its target site with better therapeutic efficiency than free drugs [74]. Two strategies are followed nowadays to exploit the magnetic functionality for stimuli-responsive drug delivery of MNPs: (i) application of 16 Chapter 1. Introduction oscillating magnetic field, leading the particles to warm their coating and releasing the drug (ii) the capacity of MNPs to also heat their environment can lead to accelerate drug diffusion [44,75]. The delivery ability of MNPs is not limited to drug, but also extends to genes [76]. Under correct design MNPs could act as a theranostic tool; thanks to their unique physicochemical and magnetic properties, as they would act as targeted drug/gene delivery carrier in a combination with MRI in order to monitor the delivery, and/or in combination with the hyperthermia to facilitate drug release and increase the therapeutic efficiency, or they would act by themselves as agent for hyperthermia and monitored by MRI. All these functions can be used simultaneously [77]. 1.4 Hurdles in nanomaterials design for successful biomedical applications Adequate design does not restrict just to chemical aspects, as important requirements in a nanomaterial for its use in medical applications have also to be considered. Instead, it requires a holistic approach where the exchange of information between medical doctors and biologists, chemists, physicists, and materials scientists will pave the way to achieve this goal. This section focuses on some important aspects that should be taken into account while designing a nanomaterial for its biomedical applications. 1.4.1 Nanomaterial core Choosing the nanomaterial core is an important issue that basically depends on the purpose of use and advantages/disadvantages considerations offered by the nanomaterial itself. As mentioned before, there are two types of nanomaterials; organic and inorganic, both are involved in many biomedical applications, therefore, we will briefly review some examples:  Quantum dots (QDs): they provide a mean for several biomedical 1.4. Hurdles in nanomaterials design for successful biomedical applications 17 applications such as in vivo imaging, drug delivery, biosensing, cell labelling, immunolabelling, can be used in fluorescence resonance energy transfer (FRET) technology and serve as ideal flourophores for ultrasensitive, multicolour and multiplexing applications in molecular biotechnology and bioengineering [36,78,79]. These applications rely on the several advantages offered by QDs, such as water solubility, broad excitation spectra, tuneable emission spectra, narrow symmetric emission spectra and high photostability [80]. However, the toxicity of some QDs is one of the biggest obstacles for their use in biological systems [81].  Superparamagnetic iron oxide nanoparticles: they offer attractive biomedical applications such as contrast agent for MRI [82], targeted drug and gene delivery system [83], agents for hyperthermia [69], bioseparation [84], immunisation [85], tissue repair [86] and detoxification of biological fluids [87]. Such a large variety of uses for an inorganic nanomaterial arises from their magnetic functionality, high field irreversibility, high saturation field, low toxicity, biodegradability, deep tissue imaging and their non-invasive tool characteristics. Thus, SPIONs can be moved or fixed by magnetic field gradients, they modify the relaxation time of neighbour protons providing strong contrast enhancement in MRI and they can convert magnetic energy into heat under the effects of an AMF in hyperthermia processes with lower adverse effects than radiotherapy or chemotherapy [88].  Liposomes: they are organic nanomaterials possessing several advantages such as amphiphilicity, biodegradability, low toxicity, ease modification and targeting potential [20]. They are, therefore, used extensively as a targeted delivery system for drugs, vaccine, anticancer and genes [89], as well as a mean for molecular imaging [90] and hyperthermia [31]. Although liposomes serve as a good model for targeted delivery, these nanomaterials are accompanied with several limitations such as lack of controlled release properties, limited drug 18 Chapter 1. Introduction loading volume, oxidation of liposomal phospholipid and poor shelf stability [91]. 1.4.2 Stability The in vitro and in vivo stability of the nanomaterial suspension is an utmost important issue. In both cases, stability is achieved through grafting or adsorption of a polymeric surfactant or other modifiers to the nanomaterial surface, forming a layer that prevents nanomaterial flocculation [92]. Moreover, the polymer coating can decrease the protein adsorption to the NPs surface. As in the case of MNPs without surface modification, and according to the pH and the ionic strength of the suspending media, nanoparticles can tend to agglomerate, which in turns has a negative impact on their in vitro and in vivo behaviour and properties. Upon intravenous injection. Agglomeration of MNPs results in increasing the nanoparticle size, which in turn affects on:  The NP biodistribution. For instance, NPs with a size more than 200 nm accumulate more in the spleen [93],  The NP internalisation into the cell. As each cell has its own optimum size for higher NPs uptake,  The subcellular fate of the NPs. Since there are several mechanisms on the cell plasma membrane that are responsible for NPs internalisation, these mechanisms differ according to NPs size [94]. For example, NPs of 60 nm size are internalised through a mechanism known as caveolinmediated endocytosis, which bypasses the endolysosomal system and ends at the cytoplasm. On the other hand, NPs with about 120 nm size are internalised through a mechanism known as clathrin-mediated endocytosis, which ends at the endolysosomal system. In some cases this is undesirable due to the harsh acidic condition that may cause acid etching of the MNPs causing a loss of magnetic properties and inducing toxicity. In contrast, presence of the nanomaterial in the endolysosomal compartements maybe favourable, as it is the case of stimuli-responsive nanomaterials, releasing their cargo (e.g. drugs) under acidic condition, 1.4. Hurdles in nanomaterials design for successful biomedical applications 19  The superparamagnetic properties of the NPs. Since the increase in size over a given value reduces superparamagnetic properties. Besides size effects, the hydrophobic surface of a NP has also a negative impact on its behaviour, since upon intravenous injection, NPs become in contact with blood that contains about 3700 types of plasma proteins. Several types of protein called opsonin, such as immunoglobulin, complement proteins, etc., are adsorbed at the hydrophobic surface of the NPs forming a protein corona, which produces signals to macrophages to come in order to eliminate the NPs from the circulation and send them to the liver, spleen or bone marrow. This process is known as opsonisation, see Fig. 1.6(a). Protein corona formation affects to the NPs circulation time, biodistribution, clearance, cellular uptake and immunogenicity. A way to resist the protein adsorption, thus increasing NPs blood circulation time, and to reduce NPs aggregation is to carry out surface modification through polymeric coating. The used polymers are either synthetic or naturally and they are either physically or chemically adsorbed to the NPs surface. The most commonly used polymer is polyethylene glycol (PEG) since it is inexpensive, versatile and is currently listed as “generally recognised as safe” (GRAS) by FDA. Addition of PEG to the NPs surface (PEGylation) increases the blood circulation time, reduces the non-specific binding of proteins Fig. 1.6(b), and reduces NPs aggregation, resulting in so called “stealth” behaviour [95]. 1.4.3 Targeting One of the factors governing NPs efficiency is their ability to specifically reach to their action site (targeting). Targeting of a nanomaterial refers to differential spatial localisation of the nanomaterial, which could be achieved through two ways: passive targeting or active targeting. Passive targeting could be accomplished by:  Direct injection of the nanomaterial into the tumour site, which is helpful in case of local cancers such as prostate, head and neck cancers developing sufficiently large tumours [21]. 20 Chapter 1. Introduction Figure 1.6: Effect of surface modification on the NP circulating time: (a) uncoated NP, the hydrophobic surface of the NP permits the adsorption of opsonin proteins (a2), which in turn facilitate the uptake of the NP by the macrophages (a3), and their accumulation in the liver (a4). (b) PEGylated NP, the PEG coating reduces the non-specific protein adsorption to the NP surface (b2), increasing the NP circulation time (b3) [95].  Enhanced permeability and retention mechanism that was discovered by Matsumura and Maeda. [96,97] and is only relevant to oncology applications. This mechanism exploits the anatomical difference between normal and tumour tissues. Tumour vasculature has endothelium discontinuities forming pores with average size ranging between 400-600 nm [98]. This allows a larger accumulation of circulating nanomaterials (with a molecular weight above 50 kDa [20]) in tumour tissues than in normal tissues with slower clearance due to lack of effective tumour lymphatic drainage [99,100]. The process is schematically represented in Fig. 1.7(a) [101]. EPR also occurs in inflammatory diseases, but with shorter retention time compared to cancer. EPR effect has been observed for a wide range of nanomaterials such as SPIONs, liposomes and micelles. EPR is associated with several limitations: (1) In large tumours, EPR is absent in the tumour centre, thus causing a decrease of nanomaterial accumulation, (2) The tumour tissue has a pressure called interstitial fluid pressure (IFP), this pressure is higher in the tumour core, diminishing toward the tumour periphery, what it leads to the flow of nanomaterials out of the tumour. These problems can be overcome in two ways: (a) Enhancing EPR effect with decreasing tumour pressure through 1.4. Hurdles in nanomaterials design for successful biomedical applications 21 co-administration of an adjuvant in addition to nanomaterial injections. Adjuvant can be such as vascular endothelial growth factor (VSGF), bradykinin, nitric oxide, prostaglandins, and transforming growth factor beta (TGFβ) receptor inhibitor. (b) Active targeted nanomaterials. Active targeting is relevant to oncology applications and other therapeutic areas. This process is achieved by direct conjugation of the nanomaterial to a site-specific targeting moiety, as shown in Fig 1.7(b). Figure 1.7: Schematic presentation for nanomaterials targeting: (a) passive targeting through enhanced permeability and retention and, (b) active targeting [101]. Targeting success depends on choosing the targeting moiety. It should fulfil several criteria such as low toxicity, abundance, high affinity and specificity toward their receptors at the target site and well suited to chemical modifications by conjugation. In addition, in order to avoid the toxicity of non-target tissues, targeted nanomaterials should bind to signature molecules that are exclusively expressed by the target site (e.g. Prostate-specific membrane antigen (PSMA)). Otherwise undesired effects can arise, as it is the case of targeting transferrin [22] and folate receptors [102]. These receptors are over-expressed by tumour cells and also are expressed to 22 Chapter 1. Introduction some degree on many types of non-to-be-targeted cells [15,16] with the result that toxic off-target effects cannot be totally eliminated. The targeting moiety could be antibodies [23] and their molecular fragments [103], proteins [22] or protein-like molecules, nucleic acid ligand (including aptamer [24]) and small molecules such as vitamins [104], peptides [105] and carbohydrates [106]. Conjugating the targeting moiety to the nanomaterial increases the localisation of the nanomaterial inside target cells. Park et al. showed that antibodytargeted liposomes (Anti-HER2 immunoliposomes containing doxorubicin) have a higher antitumour effect in compared with non-targeted counterparts (Liposomes with doxorubicin) [107]. Few years later, Kirpotin et al. disclosed that both targeted and non-targeted liposomes are similarly accumulated in the tumour tissue, and the enhanced anti-tumour effect of the targeted liposomes is due to their localisation in tumour cells, whereas, the non-targeted liposomes are in extracellular stroma or within macrophages [108]. Recently, the attention of scientists has geared toward the subcellular organelles targeting, as the efficiency of nanomaterials could depend on their localisation in a certain organelle. This is the case of gene delivery, in which the nanomaterial containing oligonucleotides as a cargo should be targeted to specific cells, breach the plasma membrane, escape from the endolysosomal compartements in order to avoid the cargo degradation under acidic conditions and then internalise to the nucleus through the nuclear pore with the help of a nanomaterial surface conjugated peptide called nuclear localising signal (NLS). Many technological trends for effective organelle targeting are emerging, such as those for targeted delivery to the nucleus [109,110], mitochondria [111], cytosol [112] and endosome/lysosome [113]. 1.4.4 Reporter nanomaterial The development of an efficient targeted nanomaterial does not entitle a positive confirmation of the site-specific delivery. Moreover, tracking the nanomaterials inside 1.4. Hurdles in nanomaterials design for successful biomedical applications 23 the cell is mandatory to explore the nanomaterial localisation in the subcellular organelles and their use for molecular imaging and biosensing. Nanomaterials by themselves can possess properties that facilitate their detection in vivo or in vitro such as fluorescent, optical or magnetic properties as in the case of quantum dots, gold nanoparticles or magnetic nanoparticles, respectively. However, several types of nanomaterials do not possess these previously mentioned properties, lacking therefore a way of detection. That is the case of polymeric nanoparticles, ceramic nanoparticles, liposomes, etc. Thus, conjugating the nanomaterial to a fluorescent probe is recommended. Fluorescent probes can be either organic such as dyes, fluorescent proteins and intrinsic fluorophores or inorganic such as lanthanides and QDs. Organic-based fluorescent dyes are widely used in research and diagnostic applications. However, they are associated with many disadvantages, such as fluorescence bleaching, the requirement of coloured matched lasers, etc. Fluorescent nanomaterials (e.g. QDs) can overcome all these disadvantages improving the fluorescent probes utility in the clinical diagnosis [114,115]. However, they can have high toxicity. Several criteria must be taken into account during the selection of the fluorescent probe, such as low toxicity, biodegradability, high photostability, high and spectrally narrow absorption and emission cross sections and ultrasensitive detection. Fluorescent-SPIONs are considered as dual reporter nanoparticles, where they could be detected through two ways, MRI and fluorescence, as shown in a study carried out by Lee et al. In this study they evaluate the stem cell labelling by fluorescent-SPIONs (in vitro) using MRI and fluorescent microscopy. In addition they showed the ability of fluorescent-SPIONs to track labelled human mesenchymal stem cells in vivo through fluorescent and MR imaging [116]. 1.4.5 Monodispersity Monodispersity means the state of uniformity. A monodispersed nanomaterial solution means that all nanomaterials in that solution have a narrow distribution of sizes. The main challenge faced by chemists during nanomaterials synthesis is 24 Chapter 1. Introduction controlling the polydispersity that has many adverse effects at different levels: (i) At the biological level, it can affect the nanomaterial breaching ability, since the size and the size distribution are important in case of plasma membrane breaching and the passage through pores such as nuclear pores. Therefore, controlling the polydispersity is crucial; otherwise different batches of the same nanomaterial might display different behaviour inside the cell. (ii) At the physical level, it could affect the quantum effects of the nanomaterial that are size-dependent. For instance, (a) a tight control of nanomaterial size and a narrow distribution of sizes are mandatory to obtain a very efficient fluorescent probes capable of emitting narrow light in a very wide range of wavelengths [14]; (b) the magnetic properties of MNP are affected by the size distribution [117]. 1.4.6 Nanomaterial physicochemical properties 1.4.6.1 Size During the nanomaterial synthesis several factors could affect the nanomaterial size such as reactant concentrations, reaction temperature and solvent conditions [2]. Moreover, it is evident that nanomaterial size is completely depending on the surface charge and the suspending medium conditions (e.g. pH, ionic strength, organic molecules). For instance, the nanomaterial size in the blood increases due to protein adsorption and formation of protein corona, as mentioned by Dobrovolskaia et al. [118]. Therefore, characterisation of the nanomaterial in its relevant solution is mandatory. The size of the nanomaterial has an impact on: (i) The nanomaterial biodistribution and circulation half-life time upon in vivo administration. Several studies proved those nanomaterials with sizes less than 5 nm are rapidly cleared from the circulation through extravasation or renal clearance [119] whereas nanomaterials with sizes larger than 200 nm are sequestered by the spleen as results of mechanical filtration and removal by reticuloendothelial system (RES) [97]. Therefore, nanomaterials with sizes ranging from 10-100 nm are optimal for long blood circulation time upon intravenous injection [120]. (ii) The nanomaterial toxicity, as the 1.7. References 31 [26] C. L. Grigsby and K. W. Leong, “Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery.,” J. R. Soc. Interface, vol. 7 Suppl 1, pp. S67–82, 2010. [27] G. Prencipe, S. M. Tabakman, K. Welsher, Z. 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[136] H. Xie, M. M. Mason, and J. P. Wise, “Genotoxicity of metal nanoparticles.,” Rev. Environ. Health, vol. 26, no. 4, pp. 251–68, 2011. [137] H. Ma, P. L. Williams, and S. A. Diamond, “Ecotoxicity of manufactured ZnO nanoparticles--a review.,” Environ. Pollut., vol. 172, no. null, pp. 76–85, 2013. 40 Chapter 1. Introduction [138] L. Ellegaard-Jensen, K. A. Jensen, and A. Johansen, “Nano-silver induces doseresponse effects on the nematode Caenorhabditis elegans.,” Ecotoxicol. Environ. Saf., vol. 80, pp. 216–23, 2012. [139] N. Tran, A. Mir, D. Mallik, A. Sinha, S. Nayar, and T. J. Webster, “Bactericidal effect of iron oxide nanoparticles on Staphylococcus aureus.,” Int. J. Nanomedicine, vol. 5, pp. 277–83, 2010. 2.1. Introduction 47 Oxidative stress: Oxidative stress is a state that reflects the imbalance between the production of reactive oxygen species and the ability of the cell to detoxify or repair it. ROS species can be subdivided into two groups: radical ROS (such as superoxide anion (O2•-), hydroxyl radical (OH•)), and non-radical ROS (such as hydrogen peroxide (H2O2)). Under normal conditions, ROS are produced by the cells as a consequence of aerobic metabolism [31]. Cells can tolerate a small and transient increase in ROS by an antioxidant defence mechanism. The antioxidant mechanism can be divided into (a) primary defence mechanism, which includes several enzymes such as superoxide dismutase, catalase, glutathione peroxidase and reductase, and (b) secondary defence mechanism, that is carried out through reduction of glutathione (GSH) [32]. Under normal conditions, more than 95% of glutathione in cells exists in the reduced form (GSH). Reduced levels of GSH will lead to a decrease of the cell capacity to clean ROS and, therefore, to allow oxidative stress. On the other hand, cells exposed to an environmental stress (such as pathogen or heat) induce a high level of ROS in response. If this high level of ROS persists for long time and cells fail to tolerate it, oxidative stress will be enhanced, resulting in oxidation of proteins, lipids (lipid peroxidation) and DNA (DNA strand breaks), as well as it will give rise to apoptosis or necrosis. Oxidations of protein and DNA have been shown to play a key role in the development of cancer, arteriosclerosis, arthritis and neurodegenerative disorders [33]. Nanomaterials favour the formation of ROS when exposed to light, ultraviolet (UV) light or acidic environment (e.g. lysosomes) or as a result of interaction with cellular components (e.g. mitochondria, redox-active proteins such as NADPH oxidase or cell surface receptors), possible mechanisms for ROS production by nanomaterials are shown in Fig. 2.4 [1,23]. Oxidative stress induced by nanomaterials enhances inflammation through upregulation of redox-sensitive transcription factors, including nuclear factor kappa B (NF-KB), and activated protein 1 (AP-1) [34]. In the case of SPIONs, ROS are induced as a consequence of their degradation in 48 Chapter 2. Cytotoxicity studies the lysosomes, Fig 2.4(a), with the consequent release of ferrous ions. These free ions can cross the mitochondrial membrane to induce ROS through Fenton reaction (Equation 2.1) [35]. H2O2+ Fe2+ -- Fe3++OH-+ OH• (Equation 2.1) Figure 2.4: Schematic illustration of the different mechanisms of ROS induction by the nanomaterials (a) existence of nanomaterial under harsh acidic conditions (e.g lysosomes) induces ROS, as a result of coating surface reactivity, metal surface exposure to acidic environment or due to leached ions (Fe 2+ , Cd 2+ ), (b) interaction of nanomaterials with mitochondria, causing mitochondria dysfunction, (c) interaction of nanomaterials with redox active protein such as NADPH oxidase, stimulating production of high amount of ROS in immune system cells, (d) interaction of nanomaterial with surface receptors, activating the intracellular signalling pathways, leading to expression of stress response genes which upregulate ROS [23]. Up to date, the link between ROS induction by SPIONs and cell toxicity remains unclear. Some studies show that IONP have the ability of ROS induction and subsequently cell damage [36], while others show that they have peroxidise-like activity which can diminish the cellular ROS levels [37–40]. The level of induced ROS depends on the total amount of surface area of internalised IONPs, and the stability of the coating against the intracellular degradation, for instance, citrate-coated IONPs have a much faster maximal ROS induction (4h) compared to dextranor lipidcoated IONPs [41]. Cytoskeleton defect: The cytoskeleton is composed of three types of filamentous proteins: microfilaments (actin filaments), intermediate filaments and microtubules. Cytoskeleton plays an important role inside the cells, as it affects to cell shape, 2.1. Introduction 49 motility, division, adhesion and connection with its environment. Furthermore, there is a relationship between cytoskeleton and endocytosis mechanism, as endocytosis develops through the reorganisation of the cytoskeleton. Therefore, the high demands imposed by the nanomaterials on the cellular endocytosis mechanism can affect the cytoskeleton network [42,43]. The association between the cytotoxicity of SPIONs through disruption of the cytoskeleton network and nanomaterial internalisation and their intracellular localisation has been described in several studies. Gupta et al. illustrated the relationship between the cytoskeleton deformation and the surface modification (coating) of SPIONs [42]. Other studies illustrate the relationship between the uncoated SPIONs internalisation by endocytosis and their intracellular localisation and cytoskeleton disorganisation [44]. Soenen et al. showed that the high intracellular concentration of nanoparticles transiently affects actin cytoskeleton and subsequently cell proliferation [45]; in another study Soenen et al. suggest that the mere physical presence of high level of IONPs enclosed in the lysosomes, typically located in the perinuclear region, diminishes the protein expression and strictly hinders the cytoskeleton network [46]. Genotoxicity and intracellular signalling alteration: Nanomaterials can induce genotoxic effects and alteration in the intracellular signalling pathways through two mechanisms: (i) Primary genotoxicity, which is related to the direct exposure to the nanomaterial such as: (a) the “nano” size enables the nanomaterial to penetrate the nucleus and directly bind to the DNA [26,47], (b) the localisation of large number of nanomaterials confined in lysosomes in the perinuclear region can prevent the cellular transcription and translation machinery [46], and (c) leaking of metal ions from lysosomes containing nanomaterials can alter protein expression through mRNA degradation. (ii) Secondary genotoxixity, which is the result of nanomaterial-cell interactions and releasing of other factors such as ROS. High level of ROS induced by nanomaterials can directly mediate DNA damage through single or double strand breaks. ROS can also alter gene expression and unregulate several transcription factors, such as the activation of redox-sensitive transcription factors, including NFKB [48]. So far, the relationship between SPIONs and induction of genotoxicity is still ambiguous. Several studies show that SPIONs have no genotoxic effect [49], neither affect stem cell proliferation [50] and gene expression patterns. In contrast, other 50 Chapter 2. Cytotoxicity studies studies show that SPIONs can induce genotoxic effects and inhibit stem cell differentiation [51]. 2.1.3 Cytotoxicity evaluation tests Many biomedical applications require the in vitro exposure of cultured cells to nanomaterials prior to in vivo translocation. Although in vitro studies allow simpler, faster and more cost-efficient assessment of defined toxicity endpoints, they do not reflect the real behaviour of nanomaterials in vivo. Therefore, validation and complementation with in vivo experiments are mandatory [11]. Up to date, a real conclusion about in vitro toxicity of nanomaterials remains ambiguous. This is due to a great variety in: (1) types of nanomaterials, (2) nanomaterial manufacturing methods, (3) nanomaterial physicochemical properties, (4) coating agent, (5) type of cell culture used, (6) variation in experiment conditions (incubation time and concentration), (7) type of assay used and (8) possible interference of the nanomaterial with the assay readout. Therefore, a direct comparison between results obtained from different studies is out of the question [23]. The assessment of nanomaterials cytotoxicity is based on several in vitro assays established for hazard characterisation of chemicals; however, nanomaterials are completely different and may interfere with the commonly used assays either through interaction with the assay components due to their high surface to volume ratio and the high adsorption capacity, or through interfering with the detection system due to their optical properties [52,53]. Several assays are used to investigate different aspects of nanomaterials cytotoxicity in vitro. (1) Cell viability assays, including determination of mitochondrial activity using MTT assay, assessing cell membrane integrity using lactate dehydrogenase (LDH), trypan blue or propidium iodide assay, determination of intracellular esterase activity using Calcein AM assay, detection of intact lysosomes using neutral red assay and detection of apoptosis using fluorescent Annexin V or Caspase substrates detection assay. Obviously, a comparison between the results from one assay with others is impossible as they measure different parameters. (2) Stress 2.2. Objectives 51 response assays, including detection of ROS; detection of any possible secondary effects in case of significant ROS induction, such as lipid or protein peroxidation, cytoplasmic calcium levels, cytoplasmic redox state (GSH detection) or DNA defects. (3) Detection of inflammatory response by measuring inflammatory markers such as the chemokine interleukin-8 (IL-8), or tumour necrosis factor α (TNF-α), IL-6, or IL1β [52]. 2.2 Objectives The aim of this chapter is to study the toxicological behaviour of our bioferrofluids in vitro through studying their effects on cell viability using lactate dehydrogenase test and the Acridine orange/Ethidium bromide assay, defining the type of cell death (apoptosis or necrosis) using DNA fragmentation assay and detection of Caspase3 activity, investigating their ability to induce oxidative stress by measuring the carbonyl content and glutathione (GSH) detection, and finally, detection of inflammasome activation by measuring interleukin 1β. In vivo toxicity studies of our bioferrofluids will be discussed in chapter 5. 2.3 Materials and methods 2.3.1 Bioferrofluid preparation and characterisation Bioferrofluids preparation and characterisation methods are the same as described in Annex I. The preparation compositions of maghemite (ɣ-Fe2O3) nanoparticles in a poly(4-vinylpyridine) matrix are summarised in Table 2.2. 2.3.2 Cytotoxicity studies All cytotoxicity experiments were performed at the laboratorio de toxicología molecular, Facultad de veterinariaUniversidad de Zaragoza, under the supervision of Professor Victor Sorribas. 52 Chapter 2. Cytotoxicity studies Table 2.2: Composition of maghemite–P4VP nanocomposites preparation for different samples. Sample P4VP (g/L) 1M FeBr3 (mL ) 1M FeBr2 (mL ) Fe/pyridine mol ratio Fe2+/ Fe3+ mol ratio Fe2O3 wt % R1 0.80 1.27 0.64 0.25 0.50 16.00 R2 0.80 2.54 1.27 0.50 0.50 27.60 R3 0.80 3.81 1.90 0.75 0.50 36.30 R4 0.80 5.08 2.54 1.00 0.50 43.20 R7 0.80 4.23 3.39 1.00 0.80 43.20 R8 0.80 4.01 3.61 1.00 0.90 43.20 2.3.2.1 Cell culture Opossum Kidney (OK) cells were grown in 75 cm2 NunclonTM Flask as described in ref. [54], in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) (Gibco-Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Primary cultures of rat aortic vascular smooth muscle cells (VSMC) were cultured in 75 cm2 NunclonTM Flask as described in ref. [55], in Minimal Eagle’s Medium (MEM) (Gibco-Life Technologies, Paisley, UK) supplemented as for OK cells. Both cell lines were incubated in a 5% CO2 incubator (lab Line) at 37 ºC with culture medium changes every second day until they became confluent. Cells were then washed twice with phosphate buffered saline (PBS), trypsinised with TrypsinEDTA 1X (Life Technologies) and re-suspended in their appropriate cell culture medium. Cells were then ready to be used in other assays. The cells were passaged in a split ratio of 1:5 and 1:3 for OK and VSMC respectively. Cell passages were between 55-57 and 6 for OK and VSMC respectively. 2.3.2.2 Cell viability assays 2.3.2.2.1 The lactate dehydrogenase assay Test principle: LDH assay is a colorimetric assay based on the reduction of 2.3. Materials and methods 53 yellow tetrazolium salt INT to a red formazan. Upon membrane damage, significant amounts of LDH are released from the cytosol of damaged cells. The LDH activity is measured in the cell culture supernatant using a Cytotoxicity Detection kit (Roche, Mannheim, Germany). The reaction is occurred in two steps: in the first step the released LDH reduces NAD+ to NADH+H+ by oxidation of lactate to pyruvate. In the second step the catalyst (diaphorase) transfers H/H+ from NADH+H+ to the yellow tetrazolium salt INT, which is reduced to red formazan Fig. 2.5 [56]. Figure 2.5: LDH activity detection reaction. Method: For the determination of cytosolic lactate dehydrogenase leakage, both cell lines were grown in 24-well plates, as described in ref. [55], until they became confluent. Cells were made quiescent for 24 hours previous to the treatments with bioferrofluids by incubating them in culture medium containing 0.5 % FCS. After 24 hours, the supernatants were aspirated out, cells were washed twice with PBS. Then, both cell lines were treated with 0.5 % FCS culture medium containing different dilutions (0, 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6) of bioferrofluids (R1, R2, R3, R4, R7 and R8). Cells treated with detergent 0.5% triton X-100 were considered as positive control Aliquots were taken from cell supernatant at different time points (0, 1, 2, 3, 4 and 5 days) followed by centrifugation at 4ºC. Then, they were processed to measure 54 Chapter 2. Cytotoxicity studies the LDH activity using Cytotoxicity Detection kit with the help of DTX-880 multimode detector system (Beckman Coulter, Indianapolis IN, USA) at 450 nm absorbance. Percentage of cell death was determined with respect to the maximal activity/absorbance obtained by positive control. The dose-response curves were plotted as the log Fe2O3 g/L versus the percentage of total LDH activity. The mean lethal concentration (LC50) was determined by non-linear regression equation using GraphPad Prism 5.0 software (San Diego, CA, USA). 2.3.2.2.2 Acridine Orange/Ethidium Bromide (AO/EB) assay For microscopic evaluation of cell death, fluorescent light microscopy with differential uptake of fluorescent DNA binding dyes (AO/EB staining) was used. Test principle: Acridine orange (AO) is a nucleic acid selective fluorescent dye that penetrates all cells and binds to DNA and RNA, making them fluorescence green and red, respectively. In contrast, Ethidium Bromide (EB) is only taken up by cells when the cytoplasmic membrane integrity is lost and stains the nucleus red. EB emission dominates over AO, therefore, live cells have a normal green nucleus, early apoptotic cells have bright green nucleus with condensed or fragmented chromatin, late apoptotic cells display condensed and fragmented red chromatin, and cells that have died from direct necrosis have a structurally normal red nucleus. Method: OK cells were seeded in 8 chamber slides (BD Falcon, Erembodegem, Belgium), and grown in their appropriate culture medium. After overnight growth, the supernatants were aspirated out and the cells were treated with aliquots of 0% FCS culture medium containing different concentrations (0, 0.007, 0.01, 0.02 g/L Fe2O3) of bioferrofluids (R1 and R8) for 24 hours. After 24 hours, the supernatants were aspirated out and the cells were washed twice with PBS, stained with acridine orange plus ethidium bromide in PBS as described in ref. [57,58]. The slides were mounted and examined rapidly under Axiovert 200M fluorescence microscopy (Carl Zeiss, Jena, Germany) after excitation at 495 nm. Green and red fluorescence were detected simultaneously using a dual band pass filter for emission at 530 nm and 610 nm. Phase contrast images of cells have been taken as well. 2.3. Materials and methods 55 2.3.2.3 Apoptosis Assays 2.3.2.3.1 DNA fragmentation Apoptosis or program cell death plays an important role in the natural renewal of cells, as well as in several different disease states. During apoptosis, cells undergo many morphological and biochemical changes different to necrosis. One of the biochemical changes is the fragmentation of the nuclear DNA, producing fragments with length varying between 180 to 200bp. In order to examine the bioferrofluids ability to induce apoptosis, deoxynucleotidyl transferasedUTP nick end labelling (TUNEL) fluorescent assay was used. Test principle: TUNEL assay is based on the incorporation of modified dUTP (e.g. fluorescein-dUTP) by the enzyme terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA. The modifications of dUTP are flurophores or small molecules, called haptens. Method: OK cells were seeded in 8 chamber slides and grown in their appropriate culture medium. After overnight growth, the supernatants were aspirated out and the cells were treated with aliquots of 0% FCS culture medium containing different concentrations (0, 0.007, 0.01, 0.02 g/L Fe2O3) of bioferrofluids (R1 and R8) for 24 hours. After 24 hours, the supernatants were aspirated out and the cells were washed twice with PBS. The cells were processed for TUNEL assay under the protocol instructions supplied with Click-iT TUNEL Alexa fluor-488 kit (Molecular Probes-Life Technologies, Paisley, UK), cells treated with DNase I were considered as Positive control. The slides were examined under the Axiovert 200M fluorescence microscopy at excitation filter of 470 ± 20 and emission filter of 525 ± 25. Phase contrast images of cells have been taken as well. 2.3.2.3.2 Caspase3 Assay Members of the caspase family proteases (especially activated caspase-3) are 56 Chapter 2. Cytotoxicity studies considered to be crucial mediators for the complex biochemical events associated with apoptosis. Therefore, apoptosis was also evaluated fluorometrically by measuring the activity of caspase-3, which is one of the most commonly used apoptosis assays. The cystein protease Caspase-3 is produced as a zymogen in the cytosol that is activated by cleavage into active caspase-3. Activated caspase-3 has substrate specificity for the amino acid sequence Asp-Glu-Val-Asp (DEVD) and cleaves several proteins leading to apoptosis. Test principle: In case of apoptosis, the elevated level of activated caspase-3 produced can be detected using EnzChek® Caspase-3 Assay kit #2 (Molecular Probes). Activated caspase-3 detection method is based on measuring the cleavage of caspase-3 substrate (DEVD) that is linked to a flourophore (Rhodamine 110), the flourophore absorbs or emits light when it is separated from the substrate. Method: OK cells were seeded in 57 cm2 Petri dishes, grown in their appropriate culture medium until they became confluent. The supernatants were aspirated out and the cells were treated with aliquots of 0% FCS culture medium containing different concentrations (0, 0.007, 0.01, 0.02 g/L Fe2O3) of R1 and R8 bioferrofluids for 24 hours. After incubation, the supernatants were aspirated out, followed by cellular wash with PBS thrice. The cells were processed for (a) protein determination using BCATM protein assay kit (Thermo scientific) and (b) caspase-3 detection under the protocol instructions supplied with EnzChek® Caspase-3 Assay kit #2. The fluorescence was measured with the help of DTX-880 multimode detector system at excitation/emission filters of 496/520 nm. This experiment was repeated 3 times in duplicate. The MannWhitney test was used for the statistical analysis of the experimental data. The results are presented as mean ± standard error of mean (SEM). Statistical significance was p < 0.05. 2.3.2.4 Oxidative stress 2.3.2.4.1 Carbonyl content measurement There is a debate over the IONPs and their ability to produce ROS. Many studies stated that IONPs have oxidative capabilities [36], while others stated that IONPs have 2.4. Results 63 The whole composition of the maghemite/polymer composite beads does not appear clearly by cryo-TEM Fig. 2.9A. Images showed maghemite nanoparticles are uniformly distributed in the solidified water matrix, surrounded by an area with a slight contrast over the background, which may correspond to the P4VP polymer that has a higher packing density than PEG polymer due to its hydrophobic character. Figure 2.7: TEM images of maghemite magnetic nanoparticles in bioferrofluids (a) R1, (b) R2, (c) R3, (d) R4, (e) R7, and (f) R8. 64 Chapter 2. Cytotoxicity studies Figure 2.8: Size distribution analysis of spherical maghemite magnetic nanoparticles (a) R1, (b) R2, (c) R3, (d) R4, (e) R7, and (f) R8. However, the presence of PEG polymer is not revealed due to a low contrast difference between the polymer and the water matrix. Nevertheless, the contour of the whole composite beads becomes apparent after surface functionalisation with antibodies Fig. 9B, or thermometric lanthanide complexes Fig. 9C. It is observed that the beads have an oval shape and contain the maghemite nanoparticles in the interior. The nanoparticles i study here in this work, however, are the basic ones without antibodies and lanthanides functionalisations. The reason for it stays in part on time constraints and, also, because these kind of functionalised nanoparticles where developed when this thesis was with its last steps. 2.4. Results 65 Figure 2.9: Cryo-TEM images of sample R8 (A), the sample R8 after conjugation with an antibody (B) or lanthanide complexes (C). Inserts show the detailed composition of the composite bead. 2.4.2 Cell Viability assay 2.4.2.1 LDH assay The cytotoxicity of these bioferrofluids was assayed in two cell lines representing epithelial (OK cells) and mesenchymal (VSMC) origins. Cytotoxicity was first evaluated as total cell death, according to the intracellular LDH released to the incubation medium, as a function of dose and time of treatment. LDH experiment has been carried out for all samples of bioferrofluids with several dilutions (0, 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6) in VSMC and OK cells at different time points (0, 1, 2, 3, 4 and 5 days). Obtained cytotoxicity data in both cell lines showed the classical sigmoidal dose-response curves when plotted as a logarithmic function of iron oxide concentration (Fe2O3 g/L) Fig. 2.10. After 1 day of treatment, VSMC showed to be sensitive (higher toxicity response) to all samples of bioferrofluids (R1, R2, R3, R4, R7 and R8) Fig. 2.10(a). In contrast, OK cells showed to be more sensitive to R1 (smaller size) in respect to other samples (R2, R3, R4, R7 66 Chapter 2. Cytotoxicity studies and R8) that show lower toxicity compared to R1 Fig. 2.10(b). Fitting the data to a sigmoidal equation by non-linear regression provided values of LC50 for each sample that were in the range of 7-16 mg/L Fe2O3 and 10-20 mg/L Fe2O3 for VSMC and OK cells respectively after 1 day of treatment. Figure 2.10: LDH activity after 1 day of treatment for all samples of bioferrofluids in both VSMC (a) and OK cells (b). The experiment was carried out during 5 successive days with the determination of LC50 for all bioferrofluids samples in both cell lines, as shown in Fig. 2.11. The data are summarised in Tables 2.4 and 2.5 for VSMC and OK cells respectively. The LC50 values for VSMC are smaller than for OK cells, and LC50 values decrease with the time in both cell lines. 2.4. Results 67 Figure 2.11: Variation of LC50 (as g/L Fe2O3) with time for all samples of bioferrofluids in VSMC (a) and OK cells (b). Table 2.4. LC50 (as mg/L Fe2O3) values for all samples of bioferrofluids in VSMC Table 2.5. LC50 (as mg/L Fe2O3) values for all samples of bioferrofluids in OK cells. Day 1 Day 2 Day 3 Day 4 Day 5 R1 6.89 5.11 3.70 2.73 1.55 R2 8.63 5.93 4.86 3.68 2.29 R4 9.44 6.72 5.37 3.93 2.50 R3 10.58 7.54 6.13 4.65 3.11 R7 13.50 8.41 6.65 5.39 3.45 R8 15.54 10.59 7.67 6.11 4.71 Day 1 Day 2 Day 3 Day 4 Day 5 R1 9.60 4.36 3.49 2.86 2.78 R2 11.53 8.72 6.33 4.20 3.57 R4 14.31 10.81 7.73 6.17 4.04 R3 15.67 12.11 9.36 7.61 5.04 R7 18.49 14.01 10.79 9.18 7.26 R8 20.11 15.60 13.17 10.31 8.87 68 Chapter 2. Cytotoxicity studies Toxicity was also analysed as a function of compositional and structural parameters of bioferrofluids. When the LC50 values after 1 day of treatment were plotted against the maghemite nanoparticle diameters, the bead hydrodynamic diameters and the iron oxide content indicated in Table 2.3, the relationships were linear in both cell lines Fig. 2.12(a), (b) and (c). The slopes of the regressions lines were positive, apparently meaning that toxicity decreases with the increasing of the diameters as well as of the iron oxide content in both cell lines. Figure 2.12: Linear correlation between LC 50 (as g/L Fe 2 O 3 ) after 1 day treatment and maghemite nanoparticles diameters (a), bead hydrodynamic diameters (b), iron oxide content (c), number of beads per litre bioferrofluids (d) and polymer/ iron oxide content ratio (e) for all samples of bioferrofluids in VSMC and OK cells. In contrast, when the same LC50 values were plotted against the number of beads per litre of suspension and the ratio of the organic component (P4VP-g-APEG) and 2.4. Results 69 iron oxide content in both cell lines (Table 2.3), the relationships were linear in both OK and VSMC and the slopes of the regression lines were negative, indicating that the toxicity is directly proportional to these two factors in both cell lines, as shown in Fig. 2.12(d) and (e). In order to confirm the relationship between the DH and the cytotoxicity on OK cells, a fixed concentration of iron oxide (0.02 g/L Fe2O3) from each sample (R1, R2, R3, R4, R7 and R8) was incubated with OK cells for 1 day. LDH activity was analysed and the results showed an inverse relationship between the hydrodynamic diameter and the cell toxicity, as shown in Fig. 2.13. Figure 2.13: Effect of bead hydrodynamic diameter on the cytotoxicity of OK cell after 1 day of treatment. 2.4.2.2. Acridine Orange/Ethidium Bromide assay Cytotoxicity was confirmed by microscopy using both phase contrast and double staining with AO/EB. Fig. 2.14 shows OK cells treated for 24 hours with R1 bioferrofluids at concentrations lower, similar and higher than the estimated LC50. Dead cells are red stained with EB, while live cells allow only the entrance of AO and therefore fluorescence green with a faint red staining in cytoplasm corresponding to RNA and lysosomes. Phase contrast illumination of the cells suggests a necrotic type 70 Chapter 2. Cytotoxicity studies of cell death with no evidence of apoptosis. Identical findings were obtained with R8 bioferrofluids as shown in Fig. 2.15. Figure 2.14: Confocal micrographs of AO/EB stained OK cells treated with different concentrations of R1 bioferrofluids for 24 hours. Faint red/orange spotty staining in Control corresponds to the intercalation of AO in RNA and lysosomal staining. Scale bar: 100 µm. Figure 2.15: Confocal micrographs of AO/EB stained OK cells treated with different concentrations of R8 bioferrofluids for 24 hours. Faint red/orange spotty staining in Control corresponds to the intercalation of AO in RNA and lysosomal staining. Scale bar: 100 µm. 2.4.3 Apoptosis assay Even if morphology of the cells did not indicate evidence of apoptosis as previously shown in AO/EB assay, two different methods were used to confirm this 2.4. Results 71 point based on the detection of both early and late apoptotic events. 2.4.3.1 DNA fragmentation (late apoptotic event) OK cells treated with different concentrations of R1 bioferrofluids showed absence of DNA fragmentation, which is indicated as a black field with no fluorescence after using a fluorescent TUNEL assay. However, positive control, as corresponds to DNase I treated cells, was fluorescent green, as shown in Fig. 2.16. Identical findings were obtained with R8 bioferrofluids, as shown in Fig. 2.17. Phase contrast images were taken to confirm the presence of the cells. Figure 2.16: Confocal micrographs of fluorescent TUNEL stained OK cells treated with different concentrations of R1 bioferrofluids for 24 hours. Positive control is DNase I treated OK cells. Scale bar: 100 µm. Figure 2.17: Confocal micrographs of fluorescent TUNEL stained OK cells treated with different concentrations of R8 bioferrofluids for 24 hours. Positive control is DNase I treated OK cells. Scale bar: 100 µm. 72 Chapter 2. Cytotoxicity studies 2.4.3.2 Caspase-3 activity detection (early apoptotic event) A fluorescent caspase-3 assay was carried out in OK cells treated with different concentrations of R1 and R8 bioferrofluids for 24 hours. Results showed no significant change in caspase-3 level compared to the control (untreated OK cells) Fig. 2.18, resulting in no evidence of apoptosis detected. Data corresponding to caspase-3 activity detection are analysed using Mann-Whitney test and are summarised in Table 2.6. Figure 2.18: Caspase-3 activity detection in OK cells treated with different concentrations of R1 and R8 bioferrofluids for 24 hours. Values represent mean ± SEM (n=6). Table 2.6. Statistical analysis data for caspase-3 activity detection in OK cells treated with different concentrations of R1 and R8 bioferrofluids for 24 hours (n=6). [Fe2O3] g/L R1 bioferrofluids R8 bioferrofluids Caspase-3 activity Mean ± SD (µM caspase/µg protein) P (MannWhitney) Caspase-3 activity Mean ± SD (µM caspase/µg protein) P (MannWhitney) 0 0.0186 ± 0.003 - 0.0186 ± 0.003 - 0.007 0.0159 ± 0.003 0.1797 0.0179 ± 0.003 0.8182 0.01 0.0164 ± 0.004 0.4848 0.0185 ± 0.005 0.9372 0.02 0.0146 ± 0.005 0.1320 0.0151 ± 0.004 0.1320 2.4.4 Oxidative stress 2.4.4.1 Carbonyl content measurement 2.5. Discussion and conclusions 79 be related to the surface charge. A study carried out by Schaeublin et al., shows that positively charged gold nanoparticles cause apoptosis, while neutral ones cause necrosis in HaCat cells [29]. Beside all the previously mentioned factors that affect the NPs toxicity, a sizedependent toxicity was also observed in both cell lines, see Fig. 2.12(a), (b), which is clearer in case of OK cells, see Fig. 2.13. Toxicity decreases as maghemite core size and bead hydrodynamic size increase, which will be explained in the next chapter by a lower uptake observed in larger nanoparticles sizes, as shown in Fig 3.9(a). A similar finding was observed by Mahmoudi et.al. who observed a decrease in the cellular toxicity of PVA coated SPIONs by increasing the hydrodynamic diameters, due to lower uptake [28]. In addition, cell death seems to be concentration dependent, as indicated by the linear correlations shown in Fig. 2.12(c), and inversely proportional to the total number of nanoparticles (total surface area) Fig. 2.12(d). The subsequent studies have been focused on OK cells only, as the preliminary in vivo studies indicated accumulation of nanoparticles in kidney. Exposure to SPIONs has been associated with apoptosis [35]. In this work we show that our SPIONs cause necrotic cell death, which is indicated by absence of the common apoptotic markers: DNA fragmentation, see Figs. 2.16 and 2.17, and caspase3, see Fig. 2.18. The relationship between SPIONs and ROS induction is not clear. Some studies show the ability of SPIONs to induce ROS [36], others show their peroxidase-like activity [40]. ROS can cause cell death through either apoptosis or necrosis, then, the link between ROS and induction of toxic effect is not clear and seems to be somehow cell type-dependent [73]. ROS induction by nanoparticles depends on several factors such as coating stability, the amount of total surface area of internalised IONPs and also the time of incubation [41,74]. After 24 hours of incubation with R1 bioferrofluids our bioferrofluids did not show any significant changes in carbonyl contents level in OK cells, see Fig. 2.19. There may be two explanations supporting this result. First, the incubation time maybe inadequate, as in this study we measured the carbonyl content level after 24 hours of incubation. Arbab et al. showed that ROS 80 Chapter 2. Cytotoxicity studies induction by SPIONs reaches its highest level after 24 hours of incubations and returns to normal after 72 hours [75]. However, Stroh et al. showed that ROS induction by SPIONs reaches its highest level after 90 minutes of incubations and disappears after 24 hours of incubation [41]. All these studies are depending on the coat stability of the nanoparticles as well as other experimental factors (e.g. cell type). Therefore, assessing the carbonyl content in time interval is mandatory. A second explanation can be in the higher stability of our coating (P4VP-g-APEG). Our maghemite nanoparticles are embedded within P4VP that is stable at acidic pH and then coated with PEG. The degradation resistance of P4VP under acidic pH protects the IONPs from degradation in the lysosmes. This explains the persistence of our nanoparticles as intact over a period of 30 days post injection in vivo without any toxic effect (chapter 5) and well as the non-oxidative stress induction. Stability of the coat and ROS induction have been the centre of several studies. Soenen et al. showed that the endosomal localisation of different coated SPIONs results in nanoparticles degradation and release of free ions that generate ROS. Thus, citrate coated SPIONs showed faster maximal ROS induction (4h) compared to other SPIONs coated with dextran or lipid [76]. Carbonyl content level results were confirmed by the normal level of GSH in nanoparticles treated cells, as shown in Figs. 2.20 and 2.21. Many nanomaterials have been reported to induce inflammation as a consequence of an elevated level of ROS or as a consequence of nanomaterial binding and activation of cell surface receptors, see Fig. 2.4(d). This leads to the activation of NF-KB, which in turns activates inflammatory genes, including genes encoding the pro-inflammatory cytokines: TNFα, IL-6 and IL-1β [34,77,78]. The pro-IL-1β is converted to activate IL-1β with the help of Caspase-1, which results from inflammasome activation. Our nanoparticles produced an elevated level of IL-1β at higher concentration (0.02 g/L Fe2O3) of R1, see Fig. 2.22, demonstrating the inverse relationship between nanoparticle size and induction of inflammatory effect. Similar results have been observed by Yang et al. using silver nanoparticles [78]. Activation of the inflammasome occurs through several stimuli, one of them being the lysosomal damage. For this reason we have carried out an assay to elucidate the 2.6. References 81 interrelationship between NPs and lysosome. Our results showed that the fate of nanoparticles seems to be their storage into the lysosomes, see Fig. 2.23. Then, when the experimental in vitro conditions lead to a massive internalisation and accumulation into lysosomes, a sudden disorder of this organelle seems to take place and necrosis of the cell arises, as shown in Figs. 2.14 and 2.15. We do not have a direct evidence for the occurrence of this mechanism, however, the accumulation of the nanoparticles in the lysosomes and the increased abundance of these organelles in the cells, see Fig. 2.23, points out to this possibility. Soenen et al. also suggested this possibility as the abundance of lysosomes in the perinuclear region could impede protein transcription, and also cause disruption in the cytoskeleton [46]. From all above we can conclude that our bioferrofluids toxicity is depending on several factors such as cell type, maghemite nanoparticle size, bead hydrodynamic size, polymer/iron ratio, Fe2+/Fe3+ ratio, concentration, total nanoparticles number , coating and charge. 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Introduction 95 such as the nanomaterial physicochemical properties (explained in section 3.1.1.1), cell type [26], cell density [26] and nanomaterial concentration. Nanomaterial surface ligands (antibodies, proteins, chemical moieties, metallic sites, polymers or surface functionalities) are the responsible to form the first link between the nanomaterial and the plasma cell membrane, resulting in internalisation of the nanomaterial inside the cell by either non-endocytic or endocytic routes of delivery, as depicted in Fig. 3.3 [3]. Figure 3.3: Schematic representation of nanomaterial-cell membrane interaction mechanisms that are responsible for nanomaterial internalisation. The parameters controlling the non-endocytic routes are marked by asterisks. In contrast, the endocytic route is controlled by specific ligand-receptor interactions [3]. 3.1.1.3.1 Non-endocytic routes of delivery The non-endocytic pathways are considered efficient tools for gene delivery, since they transport genes directly to the cytoplasm, bypassing the harshness conditions associated with the endocytic pathway such as the lysosomal degradation [27]. This direct penetration of the cell membrane is controlled by the nanomaterial surface properties (e.g. charge, hydrophobicity and roughness) which form nonspecific attractive forces to the cell membrane [3]. 3.1.1.3.2 Endocytic routes of delivery 96 Chapter 3. Nano-bio interface studies Endocytosis is a complex process with multiple mechanisms by which cells transport extracellular and plasma membrane-bound entities into the cell interior. Altogether, these mechanisms have to control the entry into the cell in a coordinated and specific manner and they play a crucial role in many cellular processes [28,29]. According to the mechanism of internalisation, macromolecules such as nanomaterials pass through different environments inside the cells that vary in harshness due to enzymatic or chemical conditions. In addition, these mechanisms define the fate of the nanomaterials inside the cell and their subcellular localisation [1]. The endocytosis mechanisms categorise as a) phagocytosis or b) pinocytosis Fig. 3.4. Figure 3.4: Different categories of endocytosis mechanisms [30]. a) Phagocytosis (cell eating): This mechanism is restricted to specialised mammalian cells, including macrophages, monocytes and neutrophils. The function of the mechanism is to engulf large pathogens or debris [30]. b) Pinocytosis (cell drinking): This mechanism is observed in all types of cells. The function of the mechanism is to internalise fluid surrounding cell [1,31]. Pinocytosis includes a handful of mechanisms distinguished by size and encapsulating vesicles composition as shown in Fig. 3.4: I. Macropinocytosis: In this mechanism, membrane protrusions are formed to collapse onto and fuse with the plasma membrane in order to transport vesicles of microns in size. This mechanism is interesting due to its avoidance of lysosomal degradation [1,30]. II. Micropinocytosis: This mechanism is including the remaining mechanisms of pinocytosis that are responsible for the internalisation of nanomaterials with sizes ranging from tens to hundreds of nanometers [1].  Clathrin-mediated endocytosis (CME): CME is the best studied 3.1. Introduction 97 mechanism and was believed for long time to be the only mechanism of endocytosis [31]. CME occurs in all types of mammalian cells, transporting essential nutrients such as lowdensity lipoprotein (LDL) and iron-laden transferrin (Tfn) into cells through their binding to specific receptors, for that CME is considered as a highly selective internalisation mechanism [32,33]. After the formation of ligand-receptor complex, a variety of proteins including cytosolic proteins called clathrins, diffuse into the plasma membrane in order to shape a coated pit around the substance to be transferred. Then, it is invaginated and pinched off to form endocytic vesicles of sizes around 120 nm. This process is dynamin-dependent [34]. CME ends with lysosomal degradation which could be: a) harmful for the nanomaterials that could not resist these harsh conditions and lack the way to escape from the endosomal compartements [27], b) beneficial for pH-sensitive delivery system, bypassing cytoplasmic drug resistance mechanisms [35,36].  Caveolae-mediated endocytosis: Caveolae are flask-shaped invaginations of the plasma membrane coated with caveolin-1, their size ranges from 50 to 80 nm [30,31]. Up to date, no studies have demonstrated the presence of caveolae able to uptake nanomaterials larger than 100 nm [31]. Caveolae mediates the internalisation of Simian virus 40 (SV40) and cholera toxin. However, other studies showed their internalisation by other endocytosis mechanisms [37,38]. This pathway facilitate the internalisation of the nanomaterial without lysosomal degradation to Golgi apparatus and endoplasmic reticulum [1].  Clathrin and caveolin-independent endocytosis: Caveolae is considered as a type of lipid raft, other types of lipid raft are small in size (40-50 nm) and diffused freely on the plasma membrane. These small rafts can be internalised within any endocytic vesicle. For example, non aggregates cholera toxins bind to rafts associated with glycolipid and internalised by clathrin-coated 98 Chapter 3. Nano-bio interface studies vesicles [30]. Currently, many methods are used to study the endocytosis mechanism of a given nanomaterial in a certain cell type, such as: a) Pharmacological inhibitors, as methyl-β-cyclodextrin (mβCD), Genistein, Cytochalasin D, potassium depletion, etc. This method is not efficient, since some inhibitors are non-specific, and may inhibit more than one mechanism. In addition, other inhibitors have a side-effect on cell physiology [31]. b) Cell expressing muted proteins and the use of siRNA. These methods have disadvantages due to their side-effect on cell physiology. Therefore, the best way to study the endocytosis mechanism is try to combine several methodologies [31]. c) Intracellular localisation of nanomaterials, using markers for cellular structures such as Lysotracker, early endosome antigen 1 (EAA1), Caveolin, etc. Measuring the colocalisation is helpful but it should be kept in mind the reasons that cause the false colocalisation depending on the way of detection [31]. 3.2 Objectives The aim of this chapter is to understand the origin of the cytotoxicity caused by our bioferrofluids described in the previous chapter. This will be achieved through several studies at the nano-bio interface level such as: studying the cellular uptake and the uptake kinetics of NPs with exploring the effect of size, cell type, NPs concentrations and time of incubation, determination of the NPs subcellular localisation and finally determination of the endocytotic mechanism by which the NPs are internalised into the cell. 3.3 Materials and methods 3.3.1 Bioferrofluids preparation and characterisation The preparation and characterisation methods of the bioferrofluids used in this chapter are described in Annex I. Their preparation conditions are summarised in Table 3.1. 3.3. Materials and methods 99 Table 3.1: Preparation conditions of maghemite polymer nanocomposites for different samples. 3.3.2 Study of the cellular uptake and the corresponding kinetic behaviour 3.3.2.1 Qualitative determination of uptake using fluorescent nanoparticles Opossum Kidney cells and vascular smooth muscle cells were seeded in 8 chamber slides (BD Falcon, Erembodegem, Belgium), and grown in their appropriate culture medium. After overnight growth of both cell lines, the supernatants were aspirated out and the cells were treated with 0.007 g/L Fe2O3 of fluorescein-labelled R8 nanoparticles (with 112 nm of hydrodynamic diameter) in serum free medium at different time points (4, 8, 12 and 24 hours). After treatment, the supernatants were aspirated out followed by cellular wash for 3 times with pre-warmed PBS. Cells were fixed with 3% (w/v) paraformaldehyde in PBS for 10 minutes at room temperature, followed by 3 times wash with PBS. After that, they were mounted and observed under Axiovert 200M fluorescence microscope equipped with ApoTome (Carl Zeiss, Jena, Germany) with excitation at 470 ± 20 nm and detection with a band pass filter at 525 ± 25 nm. 3.3.2.2 Quantitative iron detection Both cell lines (OK and VSMC) were seeded in 6-well plastic plates and grown in their appropriate culture medium. When confluent, the culture medium was aspirated out and the cells were treated with different concentrations (0, 0.001, 0.003, 0.007, 0.01 g/L Fe2O3) of R1` and R8` bioferrofluids with, respectively, 84 nm and 163 nm of hydrodynamic diameter, in serum free culture medium for 24 hours. Some samples were treated with 0.007 g/L Fe2O3 of R8` bioferrofluids at time intervals of 0, 4, 8, 12 and 24 hours. After incubation, the cells were washed several times with PBS, trypsinised, and centrifuged at 4°C for 10 minutes at 1600 rpm. The supernatant was Sample P4VP (g/L) 1M FeBr3 (mL ) 1M FeBr2 (mL ) Fe/pyridine mol ratio Fe2+/ Fe3+ mol ratio R1` 1.4 2.22 1.11 0.25 0.50 R8` 2.4 12.03 10.83 1.00 0.90 100 Chapter 3. Nano-bio interface studies discarded and the pellet was re-suspended in 400 μL Milli-Q water, using a syringe and a 25G needle. Two aliquots of 25 μL of this suspension were taken to determine the protein content, using a BCA TM Protein Assay Kit. The remaining 350 μL of this suspension was used to determine the iron content by atomic absorption. With this purpose, the suspensions were placed in Teflon tubes and left for 24 hours at 80°C to dryness. After that, 1 mL of highly purified HNO3 (>69.0%) was added, and then the samples were digested using a microwave digestor (MVS-2 Berghof). Samples were cooled and then diluted to 5 mL with Milli-Q water. Iron content was determined by graphite furnace atomic absorption spectroscopy (Varian SpectrAA with Zeeman corrector). 3.3.3 Subcellular localisation 3.3.3.1 Immunocytology Both cell lines (OK and VSMC) were seeded in 8 chamber slides and grown in their appropriate culture medium. After overnight growth, the supernatants were aspirated out and the cells were treated with 0.007 g/L Fe2O3 of fluorescein-labelled R8 nanoparticles in serum free medium for 24 hours. After 24 hours, the supernatants were aspirated out and the cells were washed 3 times with cold PBS, fixed with 3% (w/v) paraformaldehyde in PBS for 10 minutes and washed 3 times with cold PBS. They were then incubated with 20 mM glycine in PBS for 10 minutes, washed again 3 times with cold PBS, permeabilised with 0.1% (w/v) saponin in PBS for 30 minutes, washed again and incubated with the primary antibodies for 1 hour at room temperature. For the endoplasmic reticulum and early endosomes detection, an antiDerlin-1 (Sigma) antibody and an anti-EEA1 antibody (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) were used respectively. After that, the cells were washed 3 times with PBS and then incubated for 30 minutes in the dark with the corresponding Alexa fluorescent secondary antibody, followed by 3 times wash with PBS. Cells were mounted with Prolong®Gold antifade reagent with DAPI (Life Technologies, USA). For co-localisation, an Axiovert 200M fluorescence microscope equipped with ApoTome for structured illumination and Axiovision software were used. Slides were excited at 470 ± 20 nm and green fluorescence was detected with a band pass filter at 525 ± 25 nm. Red fluorescence was excited using the long pass filter at 546 ± 6 nm 3.3. Materials and methods 101 and emission at 590 nm, and DAPI was excited using the long pass filter at 436 ± 10 nm and emission at 480 ± 20 nm. Superimposition of green, red and blue fluorescence generated the final merged image. 3.3.3.2 Trackers (Lysoand Mitotrackers) Both cell lines (OK and VSMC) were seeded in 8 chamber slides, and grown in their appropriate culture medium. After overnight growth, the supernatants were aspirated out and the cells were treated with 0.007 g/L Fe2O3 of fluorescein-labelled R8 nanoparticles in serum free medium for 24 hours. After 24 hours, the supernatants were aspirated out and the cells were washed 3 times with PBS. For visualisation of the endo-lysosomal compartments, cells were incubated with 75 nM of LysoTracker® Red DND-99 (Life Technologies) for 2 hours at 37 ºC, following the manufacturer instructions. After this, the cells were washed with PBS, fixed with 3% (w/v) paraformaldehyde in PBS for 10 minutes, washed again three times, and mounted for microscopy. For mitochondria, cells were incubated with 200 nM of MitoTracker®Red CMXRos (Life Technologies) for 45 minutes at 37 ºC and then they were processed as for Lysotracker. The slides are ready for observation under Axiovert 200M fluorescence microscope equipped with ApoTome. Axiovision software was used. Slides were excited at 470 ± 20 nm, and green fluorescence was detected with a band pass filter at 525 ± 25 nm. Red fluorescence was excited using the long pass filter at 546 ± 6 nm and emission at 590 nm, and DAPI was excited using the long pass filter at 436 ± 10 nm and emission at 480 ± 20 nm. Superimposition of green, red and blue fluorescence generated the final merged image. 3.3.4 Study of endocytosis mechanism 3.3.4.1 Potassium depletion Opossum Kidney cells, were seeded in 8 chamber slides and grown as previously described. Cells were washed once with potassium-free buffer (in mM, 140 NaCl, 20 Hepes pH 7.4, 1 CaCl2, 1 MgCl2, and 5.55 D-glucose) followed by a wash with hypotonic buffer (potassium-free buffer diluted with water 1:1). After three additional 102 Chapter 3. Nano-bio interface studies washes with potassium-free buffer, cells were then incubated for 4 hours with 0.007 g/L Fe2O3 of fluorescein-labelled R8 nanoparticles in potassium-free buffer. As a positive control, different wells of OK cells were treated as above with a potassiumcontaining buffer and incubated with the same fluorescein-labelled R8 nanoparticles concentration and time in potassium-containing buffer. After incubation, the cells were washed, fixed and mounted as explained above and observed with Axiovert 200M fluorescence microscopy. Slides were excited at 470 ± 20 nm and green fluorescence was detected with a band pass filter at 525 ± 25 nm, DAPI excited using the long pass filter at 436 ± 10 nm and emission at 480 ± 20 nm. 3.3.4.2 Chlorpromazine inhibitor After cell growth in 8 chamber slides as described previously, OK cells were incubated with 10 μg/mL chlorpromazine (Sigma) for 1 hour in serum free culture medium at 37 ºC. Subsequently, cells were incubated with 0.007 g/L Fe2O3 of fluorescein-labelled R8 nanoparticles and chlorpromazine for 4 hours. As a positive control, cells were treated with transferrin from human serum conjugated with Alexa fluor®594 (Life Technologies) at a concentration of 50 μg/mL. After incubation, cells were washed, fixed, mounted and observed under Axiovert 200M fluorescence microscopy. Slides were excited at 470 ± 20 nm and green fluorescence was detected with a band pass filter at 525 ± 25 nm. Red fluorescence was excited using the long pass filter at 546 ± 6 nm and emission at 590 nm, and DAPI was excited using the long pass filter at 436 ± 10 nm and emission at 480 ± 20 nm. 3.4 Results 3.4.1 Bioferrofluids characterisation The iron contents of bioferrofluid stock samples were determined by atomic absorption in a plasma spectrometer. Concentrations of iron contents are 1.8, 8.1 and 8.1 g/L Fe2O3 for R1` bioferrofluids, R8` bioferrofluids and fluorescein-labelled R8 nanoparticles (FR8) respectively. The samples were characterised by TEM and DLS and the results are shown in Figs. 3.5, 3.6 and 3.7. Most of the nanoparticles are spherical and quite homogeneous 3.4. Results 103 in size as shown in Fig. 3.5. The size distribution analysis for the maghemite magnetic nanoparticles is shown in Fig. 3.6. The core size (mean ± SD) is 4.40 ± 0.86 and 13.08 ± 2.33 for R1` and R8` bioferrofluids respectively, the core size of FR8 is similar to that of R8`. DLS observations for each sample showed a single population of particles as represented in Fig. 3.7. The average hydrodynamic diameter for each sample is 84, 163 and 112 for R1` bioferrofluids, R8` bioferrofluids and FR8 respectively. Measurements of zeta potential of the nanoparticles suspension for all samples yielded average values close to zero. Figure 3.5: TEM images of maghemite magnetic nanoparticles in bioferrofluids (a) R1`, (b) R8`. Figure 3.6: Size distribution analysis of spherical maghemite magnetic nanoparticles (a) R1`, (b) R8`. 104 Chapter 3. Nano-bio interface studies Figure 3.7: Distribution of hydrodynamic diameters in bioferrofluid samples from DLS measurements. 3.4.2 Study of the cellular uptake and the corresponding kinetic behaviour Nanoparticles internalisation into VSMC and OK cells was studied by two ways: qualitatively using fluorescein-labelled NPs and quantitatively using non-fluorescent NPs and atomic absorption. 3.4.2.1 Qualitative determination of uptake using fluorescent nanoparticles Both cell lines (OK and VSMC) were treated with a non-toxic dose (0.007 g/L Fe2O3) of FR8 for 4, 8, 12 and 24 hours and visualised under fluorescence microscope. A significant amount of fluorescence was observed after 4 hours of incubation, which increased by increasing the incubation time up to 24 hours in both cell lines. FR8 showed a perinuclear dotted pattern of staining in both OK and VSMC Fig. 3.8. In the case of VSMC, nanoparticles were surrounded completely the nuclei (Fig. 3.8, lower panel), while inside OK cells the nanoparticles were located laterally to the nuclei (Fig. 3.8, upper panel), most likely as a consequence of the epithelial origin and differentiation of the cells. 3.4. Results 111 3.4.4 Study of endocytosis mechanism There are several methods that can be used to study the endocytosis mechanism of a given nanomaterial in a certain cell type. A method based in pharmacological inhibitors has been used in this work. 3.4.4.1 Potassium depletion Potassium depletion of cells removes the clathrin-coated pits from the cell membrane and therefore it specifically prevents this pinocytotic mechanism. OK cells were washed with potassium free buffer followed by washing with hypotonic solution and then incubated for 4 hours with potassium free buffer containing 0.007 g/L Fe2O3 fluorescein-labelled R8 nanoparticles. Results showed inhibition in the nanoparticles internalisation due to potassium depletion exposure as shown in Fig. 3.16. Therefore, this result suggests that nanoparticles are internalised following a clathrin-dependent endocytosis route. Figure 3.16: Effect of Potassium depletion on the nanoparticles internalisation by OK cells. (a) Positive control cells (b) Potassium depletion treated cells. Scale bar: 20 µm. 3.4.4.2 Chlorpromazine inhibitor In order to confirm that our nanoparticles internalised through clathrin-mediated endocytois, another inhibitor of clathrin-coated pit formation, like chlorpromazine, was used. OK cells were incubated for 1 hour at 37ºC with 10 µg/ml chlorpromazine, followed by incubation with 0.007g/L Fe2O3 fluorescein-labelled R8 nanoparticles and chlorpromazine for 4 hours. The concentration of the inhibitor was adjusted using transferrin, which is considered a ligand exclusively internalised via the clathrin 112 Chapter 3. Nano-bio interface studies coated pit pathway. Results showed that nanoparticle internalisation was completely inhibited upon chlorpromazine treatment, as shown in Fig. 3.17. Similar results were obtained using fluorescent transferrin as a positive control. These results confirm that a clathrin-mediated endocytosis route is the responsible for the nanoparticle uptake. Figure 3.17: Effect of chlorpromazine on nanoparticles uptake by OK cells. (a) cells incubated with transferrin; (b) cells incubated with transferrin and chlorpromazine; (c) cells incubated with fluoresceinlabelled R8; (d) cells incubated with fluorescein-labelled R8 and chlorpromazine. Scale bar: 20 µm. 3.5 Discussion and conclusions The ability of the nanomaterials to breach the plasma cell membrane is considering as a crucial factor for their efficacy in biomedical applications. Hence, studies pertaining to nanomaterial cellular uptake are of utmost importance. Fluorescent microscope confers the way to visualise the nanomaterials distribution inside the cell. This work has been centred to visualise the internalisation of fluorescein-labelled R8 nanoparticles (112 nm) in two cell lines (OK and VSMC). The nanoparticles were not detected after 2 hours of incubation (data not shown), but were evident after 4 hours of incubation and their internalisation increased in a time dependent manner, as shown in Fig. 3.8. These results were confirmed by quantitative measurements of the cellular iron content using acidic digestion method and atomic absorption spectroscopy, as depicted in Fig. 3.11. It was evident not only a time dependent internalisation, but also a cell type dependent internalisation. Thus, OK cells were reached their higher uptake concentration at 12 hours, as indicated in Fig. 3.5. Discussion and conclusions 113 3.11(a), while VSMC did not reach the saturation state up to 24 hours of incubation, as shown in Fig. 3.11(b). This could be explained by different saturation capacity between both cell lines. These kinetic uptake results are consistent with other studies [6,39–41]. Many factors, such as nanomaterial concentration, nanomaterial physicochemical properties, cell type, and others, influence nanomaterial uptake by the cells. In this work, we studied the effect of NPs concentration, NPs size and cell type on the NPs uptake. A concentration dependent uptake was reported for both VSMC and OK cells treated with R1 and R8 bioferrofluids, as shown in Fig. 3.9. However, in OK cells treated with R8 a slight decrease in the curve was noticed at the concentration 0.01 g/L Fe2O3, see Fig. 3.9(a), but this decrease could be neglected due to the large error bars. This finding is in agreement with previous studies [6,39,42]. The size dependent uptake was conspicuous in each cell line (OK or VSMC), since R1` (84 nm) internalised into the cells better than R8` (163 nm), as shown in Fig. 3.9. The internalised R1` was 1.78 and 1.30 folds of R8` in OK cells and VSMC incubated with a concentration of 0.007 g/L Fe2O3, respectively. This could explain the higher toxic effects associated with R1 as compared with R8 in both cell lines. The pronounced internalisation difference between R1` and R8` could be explained by the influence on their respective uptake due to different endocytosis mechanisms. The size effect on cellular uptake has been of previous concerns [4,7]. For instance, Huang et al. studied the uptake of positively charged PVP-IONPs in mouse macrophage cells using a series of samples with Dp = 8, 23, 37, 65 nm and corresponding hydrodynamic sizes DH = 32, 71, 102 and 118 nm, results showed a maximum of iron mass uptake per cell for a size of Dp =37 nm and DH=102 as compared with other ones, indicating that DH=102 nm is an optimal size for the macrophages uptake [6]. In another study on immortalised human T cells, internalisation of dextran coated aminated IONPs having similar core sizes (Dp = 6.1, 5.6 and 6.5 nm) but different hydrodynamic sizes (DH = 33, 53, 107 nm) and different IO loading (numbers of cores: 1.9, 5.2, 11.3), it was found that NPs were substantially uptaken only after a 114 Chapter 3. Nano-bio interface studies certain degree of amination and that internalisation rate was considerably higher (one order of magnitude) for DH = 107 nm nanoparticles [43].. Thus, both studies found a maximum internalisation rate for a hydrodynamic size somewhat above 100 nm. On the other hand, a comparison of cellular uptake of IONPs with Dp = 5 and 30 nm using porcine aortic endothelial cells, before and after coating with dextran or PEG, showed higher iron uptake for 5 nm NPs than 30 nm NPs when coated with dextran and the reverse for uncoated or PEG coated NPs [44]. Unluckily, it is difficult to extract conclusions from these results because data on aggregation and hydrodynamic diameters are missing. In our work, surprisingly, both Dp and DH have shown a direct impact on cell mortality and cell internalisation The cell type dependence of nanoparticles uptake was indicated in Fig. 3.10. At a concentration of 0.007 g/L Fe2O3, a 1.71 fold increases in the R1` uptake was detected for VSMC as compared to OK cells (see Fig. 3.10(a)), while 2.33 fold increases in the R8` uptake was detected for VSMC as compared to OK cells (see Fig. 3.10(b)). This different behaviour is due to different cell physiology, which might explain the high sensitivity of VSMC toward the nanoparticles (R1 and R8) comparing with OK cells. These results are in agreement with several previous studies [26,45,46]. Several mechanisms are responsible for the nanomaterial internalisation into the cells; each mechanism differs from the other in its components as well as in the internalisation route. During the internalisation, nanomaterials are exposed to different environments and conditions that vary in its harshness; this could be favourable or unfavourable to the nanomaterial depending on its purpose of use as well as on its properties. Hence, good understanding of the nanomaterial internalisation mechanisms and the factors affecting it will help to define the subcellular fate of the nanomaterial inside the cell, as well as improve the designing of an efficient drug delivery carrier. Fluorescein-labelled R8 nanoparticles were tracked in four different cellular organelles (endoplasmic reticulum, early endosomes, mitochondria and endolysosomal compartments) in both cell lines (OK and VSMC) using fluorescent antibodies and trackers with the help of fluorescent microscope equipped with ApoTome. Fluorescein-labelled nanoparticles were not found in the endoplasmic 3.6. References 115 reticulum, early endosomes, mitochondria or the nucleus, as shown in Fig. 3.12, 3.13 and 3.14. They are, however, observed inside the endo-lysosomal compartments (lysosomes and late endosomes) after 24 hours of incubation in both cell lines (OK and VSMC), as shown in Fig. 3.15. These results are in agreement with previous studies [39,45–47]. The existence of FR8 in the endo-lysosomal compartments suggests that the mechanism responsible of the nanoparticle internalisation is a clathrin-dependent endocytosis one. In order to confirm the endocytosis mechanism, pharmacological inhibitors (potassium depletion and chlorpromazine) were used. Potassium depletion of the cell blocks clathrin-dependent endocytosis by removing clathrin from the membrane, while chlorpromazine blocks this route by inhibiting Rho GTPase. Complete inhibition of FR8 internalisation was detected in OK cells upon potassium depletion treatment as well as chlorpromazine treatment, as shown in Figs. 3.16 and 3.17, indicating that clathrin-dependent endocytosis is the responsible route for the internalisation of FR8. These results are consistent with other previous studies [48,49].Mores studies are required, in order to reveal the R1 endocytosis mechanism, to give an explanation of the size dependent internalisation. Then, we can conclude that nanoparticles internalisation is dependent on many factors such as, incubation time, nanoparticles concentration, nanoparticles size and cell type. The obtained results give explanations for the caused toxicity by R1 in compared with R8 in both cell lines, as well as, explanations of the high sensitivity of VSMC toward the nanoparticles compared with OK cells. 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Introduction 127 tightly regulated by a number of inhibitors that inhibit the systematic release of thrombin and other pro-coagulant proteases. These coagulation inhibitors (anticoagulants) are: Figure 4.2: A portray of the coagulation cascade. Figure 4.3: Blood clot structure using coloured scanning electron microscope (SEM). It consists of a cross-linked fibrin meshwork (white filaments), in which erythrocytes (red) are trapped [35] . 128 Chapter 4 Haemocompatibility studies  The tissue factor pathway inhibitor (TFPI). An endogenous anticoagulant protein, a serine protease inhibitor, which inhibits further production of FXa and FIXa by the TF-FVIIa complex. The inhibition process occurs in two steps. First, the TFPI binds to and inactivates FXa; second, TFPI-FXa complex binds to and inhibits the TF-FVIIa complex. TFPI circulates in the plasma as a complex with plasma lipoproteins (80%) or free (uncomplexed) (5-20%). Lipoprotein-associated TFPI has less anticoagulant effect than the free TFPI. Free TFPI is released from endothelial cells to the circulation after heparin, non-heparin glycosaminoglycans, pentosan polysulphate, hypersulphated heparin, low-molecular weight heparin and tissue plasminogen activator administration [36].  Antithrombin (AT). A serine protease inhibitor, which is stimulated by heparin and heparin-like molecules that are present on the surface of the intact endothelial cells as heparan sulfate. It inhibits the coagulation by inhibiting FXa and thrombin [37,38].  Protein C. A vitamin-K-dependent protein, which is activated on the surface of intact endothelial cells by the binding between thrombin (a procoagulant and anticoagulant factor) and thrombomodulin (an endothelium-bound protein) into activated protein C (APC). APC with free protein S (a Vitamin-K-dependent co-factor protein) form a membrane bound complex, which can cleave factor Va to FV. Factor V acts as an anticoagulant agent that works in synergy with protein S in the degradation of FVIIIa to its inactive form (FVIII). Thus, inhibiting the coagulation process [39].  Nitric oxide, prostacyclin and ecto-ADPase. These substances are released from the intact endothelium and can cause a decrease in the platelets activity [30].  Heparan sulfate. It is a glycosaminoglycan that is attached to the surface of intact endothelial cells. It acts as a cofactor for antithrombin. 4.1. Introduction 129  Tissue-type plasminogen activator (tPA). It is released from the endothelial cells and binds to lysine residues expressed on the fibrin surface. It catalyses the conversion of plasminogen (plasma protein synthesised in the liver) to plasmin. Plasmin proteolytically cleaves fibrin into fibrin degradation products and D-dimer (fibrinolysis) [40]. 4.1.3 Coagulation screening tests There are several tests used to evaluate the coagulation state in vitro as prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and platelets count. The most common tests used are PT and aPTT, while thrombin time and fibrinogen test are not considered to be first-line screening tests although they are commonly performed. Platelets count should be checked in any case. 4.1.3.1 Prothrombin Time The activity of the extrinsic pathway is evaluated by measuring the prothrombin time, an assay that was described in 1935 by Dr. Armand Quick [29]. In this assay tissue factor-phospholipid-calcium mixture or thromboplastin is added to plateletspoor plasma. The most commonly used thromboplastin reagents are those derived from rabbit tissue [41]. Elongated PT time could be obtained in case of factors VII, X, V deficiencies and prothrombin deficiency as occurred with vitamin K-antagonist [42] or severe liver disease [43]. However, normal values of PT are detected in the case of haemophilia (factor VIII or factor IX deficiency). Hence, PT value does not reflect the coagulation process in vivo [30]. 4.1.3.2 Activated partial thromboplastin time The activity of the intrinsic pathway is evaluated through measuring the activated partial thromboplastin time; a modified assay from partial thromboplastin time that was discovered by Drs. Langdell, Wagner and Brinkhous in 1953 [29]. In this assay non-physiological, negatively charged compounds (e.g., celite, kaolin, or ellagic acid suspended in a phospholipid mixture) are added to platelets-poor plasma [44]. Presence of such compounds (contact system activator) activates a series of reactions in a descending order ended with the formation of thrombin. Prolonged aPTT could be obtained in case of factors XII, XI, IX, VIII and V deficiencies, prothrombin 130 Chapter 4 Haemocompatibility studies deficiency, liver disease, haemophiliaand the presence of anticoagulant agents such as heparin, argatroban, bivalirudin and lepirudin. 4.1.3.3 Thrombin time This test is used in order to evaluate the efficiency of the common pathway. It is not considered to be a first-line screening test. In this test a bovine or human thrombin is added to platelets-poor plasma, which in turns converts fibrinogen into fibrin. The time for fibrin clot formation is then recorded. Thrombin time is similar to another time called “Reptilase time” (RT) which is more frequently used, but in this test a snake venom activator is added instead of thrombin. The thrombin time is sensitive to heparin while the reptilase time is not. Elongation of TT is associated with amyloidosis, fibrin degradation products, fibrinogen deficiency/abnormality, hypoalbuminaemia and the presence of inhibitors as heparin [45–47]. 4.1.3.4 Fibrinogen test This test is used to detect abnormal bleeding or coagulation disorder. A high level of fibrinogen is associated with several disorders such as inflammation, cardiovascular diseases, strokes and cancer. However, low fibrinogen level increases the risk of bleeding and it is associated with several disorders such as haemodilution, liver diseases and inherited deficiencies (e.g. Hypofibrinogenaemia, afibrinogenaemia and dysfibrinogenaemia). Several methods are used to measure the plasma fibrinogen level such as a) Blombäck and Blombäck method, b) clotting rate assay according to von Clauss, c) immunoassays, including radial immunodiffusion (RID) according to Mancini et al., ELISA or nephelometric, d) total amount of clottable fibrinogen by means of turbidimetric assay according to Ellis and Stransky, e) Chromotime System, and f) prothrombin time-derived fibrinogen assay on ACL coagulometer [48–50]. 4.1.3.5 Platelets count Platelets count is often a part of complete blood count (CBC) assay. Abnormalities in platelets account are associated with bleeding disorders and other bone marrow diseases as leukaemia. 4.2. Objectives 131 4.1.4 Haemolysis Haemolysis is the breakdown of red blood cells, which leads to the leaking of haemoglobin and intracellular ingredients such as potassium into the plasma. Haemolysis can lead to life-threatening conditions such as anaemia, jaundice, hypertension, arrhythmia and renal failure [51]. Severe haemolysis could be detected visually with naked eyes. However, this method is unreliable in case of mild haemolysis detection, since the presence of bilirubin in serum may impair the ability to detect haemolysis visually, especially in neonatal samples in which elevated bilirubin concentration commonly happens. Mild haemolysed samples (serum free haemoglobin in the range between 0.3 and 0.6 g/L) could be detected by semiquantitative spectrophotometer measurement at wavelength 400-800 nm. Oxygenated haemoglobin is detected by two absorption peaks at the wavelengths 540 and 580 nm Fig. 4.4 [52]. In general, absorbed haemoglobin peak at 580 nm is preferable because it is a bit higher. Significant haemolysis is associated with decreasing in erythrocytes, haemoglobin and haematocrit [53]. Figure 4.4: Light absorption spectra of human fetal and adult oxygenated haemoglobin. Absorptivity is expressed in L. mmol -1 .cm -1 [52]. 4.2 Objectives The aim of this chapter is to study the in vitro toxicity of our bioferrofluids (P4VP-gAPEG bioferrofluids) and its components in blood, through investigating their effect on the coagulation process by measuring the prothrombin time and activated partial thromboplastin time, studying their effect on blood cells through measuring the blood cell count and detection of haemolysis by measuring the free haemoglobin. In 132 Chapter 4 Haemocompatibility studies addition, this chapter explores the effect of surface coating of the nanoparticles on the blood haemostasis using two bioferrofluids, one is coated with dimercaptosuccinic acid and the other is coated with dimercaptosuccinic acid with short-chain diamine PEG. 4.3 Materials and methods 4.3.1 P4VP-g-APEG coated bioferrofluids preparation and characterisation Bioferrofluids preparation and characterisation methods are the same as described in Annex I. The preparation conditions of the maghemite polymer nanocomposites used in this chapter are summarised in Table 4.1. Table 4.1: Preparation conditions of maghemite polymer nanocomposites for different samples. Sample P4VP (g/L) 1M FeBr3 (mL ) 1M FeBr2 (mL ) Fe/pyridine mol ratio Fe2+/ Fe3+ mol ratio H 0.4 1.584 0.794 The prepared P4VP-g-APEG bioferrofluid was further purified by centrifugation at 196.000 G for 30 minutes and re-dispersed in PBS by ultrasounds. The dispersion was filtered through a sterile 0.22 µm membrane filter to obtain P4VP-g-APEG bioferrofluids. 4.3.2 DMSA-bioferrofluids and DMSA-PEG-(NH2)2-bioferrofluids preparation and characterization These bioferrofluids were supplied by Prof. Puerto Morales, from the Instituto de Ciencia de Materiales de Madrid, as part of an ongoing collaboration. The synthesis was performed in three steps: 1) synthesis of magnetite nanoparticles, 2) surface modification with DMSA and 3) PEG conjugation and dispersion in water. Magnetite nanoparticles were obtained via thermal decomposition of an iron coordination complex as a precursor to ensure nanoparticle homogeneity in 4.3. Materials and methods 133 size and shape following the method reported by Sun and co-workers [54,55]. Particle size and shape were studied using a 200 keV JEOL-2000 FXII microscope. A drop of a dilute magnetic nanoparticle suspension in hexane was placed on a carbon coated copper grid and dried at 50 ºC. Size distribution was determined from TEM micrographs through manual measurement of more than 200 particles and data were analysed with Gwyddion 3.25 software to obtain the mean size and standard deviation by gaussian fitting. Particles were coated with meso-2,3-dimercaptosuccinic acid (DMSA) by a ligand exchange process to remove oleic acid, after which a short-chain diamine PEG (PEG-(NH2)2) was covalently bound to the nanoparticle surface via 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) activation of the carboxylic acids. Colloidal properties of 0.5 mM Fe nanoparticle suspensions in water were characterised by dynamic light scattering using a Nanosizer ZS (Malvern). ZAverage values in intensity at pH 7 were used as the mean hydrodynamic size. The polydispersity degree index was calculated by dividing the standard deviation by the mean size. The Z potential was measured in a 0.01 M KNO3 solution. Other properties of DMSA-bioferrofluids and DMSA-PEG-(NH2)2-bioferrofluids have been described in detail in previous publications [56,57]. 4.3.3 Coagulation studies All haemocompatability experiments were performed at the hospital Clinico Universitario Lozano Blesa, Zaragoza, under the supervision of Professor Martín Gutierrez, Doctor Rosa Cornudella and Doctor José Antonio Moreno. 4.3.3.1 Control plasma Blood samples were obtained from healthy human volunteers. Samples were collected in citrate (0.129 M) vacutainer tubes. The samples were centrifuged at 3500 rpm to obtain platelets-poor plasma (PPP). The plasma was processed for the coagulation studies of PT and aPTT, using PT and aPTT HemoSILTM reagents and measured by the coagulometer TOP-ACL from IL-Instrumentation. The results were within the reference limits (9-14 s. and 23-37 s. respectively). 4.3.3.2 PPP treated with bioferrofluids 134 Chapter 4 Haemocompatibility studies PPP samples were mixed with different concentrations of bioferrofluids (P4VP-gAPEG bioferrofluids, DMSA bioferrofluids and DMSA-PEG-(NH2)2 bioferrofluids) and processed for the measurement of PT and aPTT. 4.3.3.3 PPP treated with bioferrofluids coating components To study the effect of the coating on the coagulation, PPP samples were mixed with different concentrations of APEG, P4VP-g-APEG, DMSA, and PEG-(NH2)2and processed for the measurement of PT and aPTT. 4.3.4 Complete blood counts studies 4.3.4.1 Control blood Blood samples were obtained from healthy human volunteers. Samples were collected in EDTA K3, 1.8 mg/mL vacutainer. The blood samples were processed for CBC studies using a Coulter LH 780 analyzer from Beckman Coulter. 4.3.4.2 Blood treated with bioferrofluids, and coating materials Blood samples were mixed with different concentrations of the investigated materials (P4VP-g-APEG bioferrofluids, DMSA bioferrofluids, DMSA-PEG-(NH2)2 bioferrofluids, APEG, P4VP-g-APEG, DMSA, and PEG-(NH2)2) and processed for blood cell counting. 4.3.5 Haemolysis studies 4.3.5.1 Control blood Blood samples were obtained from healthy human volunteers. Samples were collected in Lithium heparin 17 UI/mL vacutainer tubes. The plasma free haemoglobin was analysed after blood centrifugation at 2500 rpm for 5 minutes using a double beam spectrophotometer Analytic Jena –Specord 205 with wavelength range between 500 – 630 nm. 4.3.5.2 Blood treated with bioferrofluids Whole blood was mixed and incubated with different concentrations of bioferrofluids (P4VP-g-APEG bioferrofluids, DMSA bioferrofluids, and DMSA-PEG- 4.4. Results 135 (NH2)2 bioferrofluids) for 5 min, then centrifuged at 2500 rpm for 5 min and the plasma processed for the measurement of the free haemoglobin. 4.4 Results 4.4.1 P4VP-g-APEG bioferrofluids characterisation The iron content of bioferrofluid stock sample was determined by atomic absorption in a plasma spectrometer. Concentration of iron content is 7.78 g/L Fe2O3. The sample has 13.36 g/L of P4VP, 12.03 g/L of APEG(200) and 1.34 g/L of APEG(1000)-COO-, and the necessary amounts of phosphate, sodium, potassium and chloride ions for a standard PBS solution of pH=7.40 and I = 0.15 M. The sample was analysed by TEM and DLS and the results are shown in Figs. 4.5 and 4.6. TEM images show iron oxide nanoparticles uniformly distributed in a continuous polymer film (insert in Fig. 4.5). Most of the iron oxide nanoparticles are spherical, as shown in Fig. 4.5. A size distribution analysis for the maghemite magnetic nanoparticles is shown in Fig. 4.6(a). The core size (mean ± SD) is 9.1 ± 2.1 nm. Figure 4.5: TEM images of maghemite magnetic nanoparticles in P4VP-g-APEG bioferrofluids. The inset shows the sample at lower magnification. 136 Chapter 4 Haemocompatibility studies DLS observations show a single population of particles with an average hydrodynamic diameter of 80 nm, as shown in Fig. 4.6(b). Measurements of zeta potential of the nanoparticles suspension yielded average values close to zero. The structures of components of the nanoparticles shell are shown in Diagram 1. The reaction of P4VP and APEG is a Michael addition involving the acrylate double bond and the nitrogen of the pyridine that becomes quaternised and therefore positively charged in this way. The structure of the P4VP-g-APEG graft copolymer is comb-like. The backbone is a polyethylene chain with pyridine side groups. The APEG chains are linked to some of the pyridine groups by N-C bonds between the nitrogen of the pyridine and the β-carbon of the acrylate PEG ester, thus hanging perpendicular to the copolymer backbone as depicted in Diagram 1. Therefore the N of pyridine groups linked to APEG chains are positively charged and the copolymer is cationic. Figure 4.6: P4VP-g-APEG bioferrofluids analysis: (a) Size distribution of spherical maghemite magnetic nanoparticles; (b) Distribution of hydrodynamic diameters in bioferrofluid sample from DLS measurements.