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REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES AND MAGNETIC FIELDS AND MAGNETIC FIELDSAND MAGNETIC FIELDS AND MAGNETIC FIELDS Beatriz Romartínez Alonso Directors Final Master Project REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES AND MAGNETIC FIELDS AND MAGNETIC FIELDSAND MAGNETIC FIELDS AND MAGNETIC FIELDS Beatriz Romartínez Alonso Directors : Gerardo F. Goya and M. Pilar Calatayud REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS REMOTE GUIDING OF NEURAL CELLS USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES USING MAGNETIC NANOPARTICLES AND MAGNETIC FIELDS AND MAGNETIC FIELDSAND MAGNETIC FIELDS AND MAGNETIC FIELDS Pilar Remote guiding of neural cells using magnetic nanoparticles and magnetic fields ABSTRACT Nerve regeneration, as a treatment of traumatic nerve injuries or degenerative diseases, has been pursued along the last decades, stimulating both basic and applied research. The strategies for nerve regeneration have been boosted recently by the consolidation of nanotechnology, allowing innovative approaches through physical or chemical guidance of axonal re-growth across the nerve gap after severe injury. As an example, many guidance therapies are based on nano-patterned scaffolds that serve as “nerve guidance channels” providing an effective conduit during the nerve regeneration process. These recent advances have provided new therapeutic possibilities as alternatives to established surgical techniques. The present work has been developed within the framework of a novel, minimally invasive methodology for physical axon guidance based on the use of magnetic nanoparticles (MNPs) and magnetic fields (H). This strategy is based on the hypothesis that the neural cells can, under the application of an externally produced tensile force, guide the process of neurite outgrowth and axon elongation along the desired direction imposed by this force. The central goal of this work has been to demonstrate that magnetic nanoparticles (MNPs) can be used to generate these tensile forces under the effect of an external magnetic field gradient, and these forces can in turn provoke the orientation of neurites along the magnetic field direction. The MNPs used for this purpose were polyethyleneimine-coated Fe 3 O 4 nanoparticles (PEI-MNPs) with sizes of 25 ± 5 nm and good magnetic response. Neuron-like PC12 cell line was selected for in vitro experiments. PEI-MNPs showed low toxicity effects on PC12 cells. Nanoparticle uptake quantification was carried out via iron-thiocyanate complex colorimetric assay. The intracellular distribution of the PEI-MNPs was analyzed by TEM and dual-beam (FIB/SEM) techniques. These analyses revealed the coexistence of both fully incorporated PEI-MNPs and partially-internalized PEI-MNPclusters crossing the cell membrane. The changes in morphology and cytoskeletal structure of cells exposed to PEI-MNPs were investigated using optical and confocal microscopy .When PEI-MNPs loaded PC12 cells were exposed to a static magnetic field (0.020 T), neurites outgrowth of PC12 cells was preferentially aligned to the direction of the magnetic force. An increment of the neurite length was also observed. Remote guiding of neural cells using magnetic nanoparticles and magnetic fields We demonstrated that the produced re-direction of the neurites has its origin in the magnetic force acting on the MNPs previously loaded into the neurites. Furthermore, an additional effect of the incorporated MNPs has been observed, i.e., that the MNPs can stimulate the neurite outgrowth process, opening the possibility of a two-fold therapy: remote orientation and stimulation of axonal growth. These results open the possibility of non-invasive, multifunctional therapies for nerve injury based on these MNPs and external magnetic fields. Key words: Neurite outgrowth, Magnetic nanoparticles, Neuronal differentiation, Magnetic field, Regeneration. Direccionamiento neuronal remoto usando nanopartículas y campos magnéticos RESUMEN La regeneración del sistema nervioso, como tratamiento para las lesiones nerviosas traumáticas o enfermedades degenerativas, ha sido una idea perseguida a lo largo de décadas. El surgimiento de la nanotecnología ha permitido nuevas estrategias para la regeneración nerviosa, a través de enfoques innovadores basados en materiales nanoestructurados. Varias terapias de guiado neuronal se basan en estos ‘andamios’ nano-estructurados que sirven como "canales de guía de nervios " que proporcionan un conducto eficaz durante el proceso de regeneración del nervio. El presente trabajo se ha desarrollado en el marco de una nueva metodología, mínimamente invasiva, para el guiado físico de neuritas y axones basada en el uso de nanopartículas magnéticas (NPM) y campos magnéticos aplicados remotamente. Esta estrategia se basa en la hipótesis de que las células neuronales pueden, bajo la aplicación de una fuerza de tracción producida y aplicada externamente, guiar el proceso de crecimiento de las neuritas y el alargamiento del axón a lo largo de la dirección impuesta por dichos campos magnéticos. El objetivo central de este trabajo ha sido demostrar que las nanopartículas magnéticas (NPMs) pueden utilizarse para generar estas fuerzas de tracción bajo el efecto de un gradiente de campo magnético externo, y estas fuerzas puede a su vez provocar la orientación de las neuritas a lo largo de la dirección del campo magnético. Las NPMs utilizadas están compuestas por un núcleo magnético de Fe 3 O 4 recubierto por polietilenimina (PEI-NPMs), con tamaños de 25 ± 5 nm. Células de la línea PC12 fueron seleccionadas como modelo neuronal para los experimentos in vitro. Las PEINPMs mostraron una baja toxicidad en células PC12, y su cuantificación se llevó a cabo a través de ensayos de absorción colorimétrica mediante un complejo de hierrotiocianato. El estudio de la distribución intracelular de las NPMs mediante técnicas de doble haz (FIB / SEM) y TEM reveló la coexistencia de aglomerados de NPMs internalizados en el espacio intracelular, junto con otros aglomerados parcialmente internalizados, es decir, atravesando la membrana celular. Cuando las células PC12 cargadas con PEI-NPMs fueron expuestas a un campo magnético estático se observó que el crecimiento de las neuritas ocurría preferencialmente en la dirección de la fuerza magnética externa aplicada. Simultáneamente, se observó un incremento Direccionamiento neuronal remoto usando nanopartículas y campos magnéticos estadísticamente significativo en la longitud promedio de las neuritas cuando éstas se encontraban cargadas con NPMs y sometidas al campo magnético externo. Hemos demostrado que la orientación de los axones producida tiene su origen en la fuerza magnética que actúa sobre las NPMs previamente incorporadas a las neuritas. Por otra parte, se ha observado un efecto adicional de las NPMs incorporadas, específicamente que las NPMs pueden estimular el proceso de crecimiento de las neuritas, abriendo la posibilidad de una terapia bi-funcional: la orientación remota en conjunto con la estimulación del crecimiento axonal. Estos resultados abren la posibilidad de nuevas terapias multifuncionales no invasivas para lesiones nerviosas severas, mediante el uso de NPMs y de campos magnéticos externos. Palabras clave: Crecimiento de neuritas, Nanopartículas magnéticas, Diferenciación neuronal, Campo magnético, Regeneración. Index INDEX I. INTRODUCTION ........................................................................................................... 1 1. Nanotechnology and Nanomedicine .................................................................... 1 2. Magnetic nanoparticles ........................................................................................ 1 2.1. Physical properties: nanomagnetism and superparamagnetism ..................... 3 2.2. Iron oxide nanoparticles ................................................................................. 4 3. Magnetic nanoparticles for nerve regeneration ................................................. 5 3.1. Biochemistry of neural cell differentiation and nerve regeneration ............... 5 3.2. Axon growth and guidance ............................................................................. 6 3.3. Effects of MNPs in nerve regeneration .......................................................... 8 3.4. Effects of a magnetic field in nerve regeneration and remote guidance......... 9 II. OBJECTIVES ................................................................................................................ 11 III. MATERIALS AND METHODS ................................................................................. 12 1. Synthesis of PEI coated Fe 3 O 4 nanoparticles (PEI-MNPs) and fluorescent PEI-Fe 3 O 4 nanoparticles. ...................................................................... 12 2. Physicochemical characterization of magnetic nanoparticles ........................ 13 2.1. Transmission electron microscopy (TEM) ................................................... 13 2.2. Zeta potential ................................................................................................ 13 2.3. Magnetic characterization ............................................................................. 13 3. In-vitro experiments ............................................................................................ 14 3.1. PC12 Cell culture .......................................................................................... 14 Index 3.2. Quantification of uploaded PEI-MNPs in PC12 cells .................................. 14 3.3. SEM and Dual beam (FIB/SEM) analysis of PC12 cells incubated with PEI-MNPs ....................................................................................................... 15 3.4. Immunocytochemistry assay in PC12 cells: actin and tubulin fluorescent staining .................................................................................................. 15 4. Neurites orientation and outgrowth: experimental set-up ............................. 16 5. Statistical analysis ............................................................................................... 19 IV. RESULTS ...................................................................................................................... 20 1. PEI coated Fe 3 O 4 nanoparticles: synthesis process and physical characterization ......................................................................................................... 20 2. Neuronal (PC12) cells incubated with PEI-MNPs .............................................. 22 2.1 Biological effects: cytotoxicity .......................................................................... 22 2.2. Quantification of uploaded PEI-MNPs in neural cells ..................................... 23 2.3. Biodistribution of PEI-coated MNPs in neuronal cells .................................... 24 2.4. Immunofluorescence analysis of neuronal cell cytoskeleton ........................... 29 3. Control of the orientation of growing neuronal processes in PC12 cells ....... 34 V. DISCUSSION................................................................................................................ 39 VI. CONCLUSIONS AND FUTURE PERSPECTIVES .................................................. 45 VII. REFERENCES .............................................................................................................. 46 VIII. ANNEX .......................................................................................................................... 51 Index INDEX OF FIGURES AND TABLES Figure 1. Differences between bulk ferromagnetic materials and particles with size bellow D C ................................................................................................................ 4 Figure 2. Neuronal regeneration in the PNS. ................................................................... 6 Figure 3. Nerve regeneration mediated by MNPs. ......................................................... 10 Figure 4. Chemical structure of polyethyleneimine (PEI 25 kDa) and Alexa Fluor® 488 .................................................................................................................. 13 Figure 5. Representation of the magnetic field applied to the PC12 cultures. ............... 17 Figure 6. Representation of the magnetic field applied to the PC12 cultures and image of the culture support. ....................................................................................... 18 Figure 7.TEM and HRTEM images of PEI-MNPs, and histogram showing the size distribution of PEI-MNPs ............................................................................................ 20 Figure 8. Z-potential values vs. pH for PEI-coated magnetic nanoparticles. ................ 21 Figure 9. Magnetization curves of PEI-MNPs ................................................................ 22 Figure 10. Total amount of iron content per cell at different concentration of PEIMNPs added. ................................................................................................................ 24 Figure 11. Cellular localization of PEI-MNPs ............................................................... 25 Figure 12. Scanning electron microscopy image of a single PC12 cell incubated for 24 hours with 10 µg/mL PEI-MNPs, showing the presence of PEI-MNPs agglomerates (bright spots) on the cell membrane and on the growth cone. .............. 26 Figure 13. SEM images of PC12 cells (MNP + H - ) cross-sectioned by FIB after 24 hours incubation at 10 µg/mL of PEI-MNPs and for 4 days of differentiation. .......... 27 Figure 14. SEM images of PC12 cells (MNP + H + ) cross-sectioned by FIB after 24 hours incubation at 10 µg/mL of PEI-MNPs and for 4 days of differentiation. .......... 28 Figure 15. Phase contrast images of PC12 cells: ........................................................... 30 Index Figure 16. Phase contrast images of PC12 cells ............................................................ 31 Figure 17. Immunofluorescence images of differentiated PC12 cells 4 days ................. 32 Figure 18. Immunofluorescence images of differentiated PC12 cells 4 days after treatment with f-PEI-MNPs in the presence of magnetic field (MNP + H + ). ................ 33 Figure 19. Representation of the results obtained in each condition by using “Image J”. ................................................................................................................... 35 Figure 20. Representation of the results obtained in the cells treated with PEIMNPs and magnetic field (MNP + H + ) by using “Image J” ......................................... 36 Figure 21. Number of neurites (normalized with respect to the control) as function of the angles ................................................................................................... 37 Figure 22. Analysis of neurite length distribution 4 days after the induction of differentiation ............................................................................................................... 38 Figure 23. Nerve regeneration mediated by MNPs. ....................................................... 39 Figure A1. SEM images of PC12 cells (MNP + H + ) cross-sectioned by FIB after 24 hours incubation at 10 µg/mL of PEI-MNPs and for 4 days of differentiation ........... 51 Figure A2. Phase contrast images of PC12 cells ........................................................... 52 Figure A3. Phase contrast images of PC12 cells ........................................................... 53 Figure A4. Immunofluorescence images of differentiated PC12 cells 4 days control (a) and after treatment with f-PEI-MNPs (b) .................................................. 54 Figure A5. Immunofluorescence images of differentiated PC12 cells 4 days after treatment with f-PEI-MNPs in the presence of magnetic field (MNP + H + ). ................ 55 Table 1. Effect of PEI-MNPs on PC12 cell line. ............................................................ 23 Introduction 7 circuitry in the developing brain [30], and for the re-wiring of pathways that are damaged from injury or disease [31]. Growth cone behaviors such as advancing, retracting, turning and branching are regulated by dynamic reorganization of actin filaments and microtubules, which in turn are linked to the receptors for molecules called guidance cues, such as cell adhesion molecules (CAMs: integral membrane proteins mediating adhesion between growing axons and eliciting intracellular signaling within the growth cone), developmental morphogens, growth factors (NGF), neurotransmitters, etc. Growth cones integrate complex cues which vary in both time and space, and how all of these pieces fit together is less understood. Most axon guidance receptors activate signal transduction cascades that ultimately lead to reorganization of the cytoskeleton and adhesive properties of the growth cone, which together underlie the motility of neural cells. Recent progress indicates that focal adhesions may function as the biomolecular integration point of growth cone signaling and response [32]. Focal adhesions (FAs) are integrin-based adhesions whose stability determines the initiation and extension of neurites in differentiating neurons [33, 34]. Integrins comprise a large family of cell adhesion transmembrane proteins that mediate interactions between the extracellular environment and the cytoplasm. Integrins regulate many aspects of cell behavior including cell death, proliferation, migration, and differentiation and are the predominant molecular transducers of force [35]. Neurites are long cellular protrusions off the cell body that emerge when differentiation starts. The emerging neurites grow and find their way toward specific targets in the body following the guidance cues, so, eventually, they are responsible of the migration and polarization of the differentiating neurons. The maturation of FAs likely plays an essential role in sensing local substrate information and coordinating the morphometric changes that characterize the establishment of cell polarity during neuronal differentiation [34, 36]. Goals of neural tissue engineering for nerve injury include trying to find a way to guide axons with cues to achieve directed neuronal growth. Engineered MNPs opens their potential for both positioning and guiding neural cells in response to an external magnetic field [20, 37]. In particular, the approach of using MNPs as an alternative to Introduction 8 surgical nerve grafting for the treatment of severe peripheral nerve injuries is one of the most appealing applications. 3.3.Effects of MNPs in nerve regeneration Kim and collaborators reported in 2011 that iron oxide nanoparticles can enhance neurite outgrowth [37]. Neural cells exposed to both iron oxide nanoparticles and nerve growth factor (NGF) synergistically increased the efficiency of neurite outgrowth [37]. They proposed that their results are due to the activation of cell adhesion molecules that are associated with cell-matrix interactions through iron. Iron oxide nanoparticles upregulate the expression of a neural specific marker and a cell adhesion protein (integrin). Thus, they hypothesized that iron oxide nanoparticles can affect cell-substrate interaction and regulate cell behaviors. These results are in agreement with the information presented in the previous part that expose that cell adhesion molecules regulate cell differentiation, proliferation, migration and survival by interacting with extracellular molecules, such as integrin which is one of the adhesion molecules. If iron oxide nanoparticles stimulate focal adhesion of neurites, neurites outgrowth would be improved. Recently, it has been discovered that iron oxide nanoparticles can attenuate oxidative stress in a neutral pH environment in vitro and, in combination with an external electromagnetic field, they can also facilitate axon regeneration [38]. The primary injury caused by nerve transaction is generally followed by secondary damage that results in a number of morphological, physiological, and biochemical changes, including inflammation, disruption of neurological pathways, demyelination, and cell death at the site of the lesion [38]. Cyst formation and robust infiltration of fibroblast result in formation of fibrous scar tissue that creates a physical barrier, preventing any severed axons or collateral branches of uninjured axons from growing past the area of the lesion. The free radical scavenging properties of iron oxide NPs can reduce the formation of fibrous scar tissue or secondary damage which is positively correlated with functional recovery [38]. Iron oxide nanoparticles have the potential to promote regeneration and axon growth in presence of an external magnetic field, thereby helping to bridge the gap at the injury site [39]. Retention and accumulation of iron oxide NPs in cells can be enhanced by an Introduction 9 external magnetic field , providing mechanical tensile forces that may cause axonal sprouting [39]. An external magnetic field can magnetize nanoparticles such that their beneficial effects can be focused to the site of injury. An external magnetic field also can noninvasively create intense rapid electric fields in deep underlying structures. If the current created is of sufficient amplitude and duration such that depolarization occurs, neural tissue is stimulated. Magnetic stimulation provides a microenvironment conducive to neural repair by stimulating release of neurotrophic factors that promote migration of glial cells to the site of the lesion and decrease apoptosis after neuronal injury [40]. Thus, combination of these effects (iron oxide NPs and magnetic fields) may reduce the secondary damage and facilitate of axonal and neuronal regeneration, by reducing the lesion volume and due to the neuroprotective effect against oxidative stress [38]. 3.4.Effects of a magnetic field in nerve regeneration and remote guidance It is well documented that cells can respond to the mechanical properties of their environments, including stiffness [41], shear flow [42], and mechanical stretch [41]. The process of mechanotransduction, which is how cells convert physical force into a biochemical signal, has been shown to occur through integrins, as well as through mechanosensitive ion channels [43] and G-protein coupled receptors [44]. In addition, focal adhesion sites have the ability to strengthen when forces are applied to them. So, mechanosensing has been hypothesized to play a role in cellular migration. It has been suggested that mechanical forces on cellular membranes are capable of rearranging membrane components such as integrin complexes. Focal adhesion structures are capable of changing localization, composition and cellular behavior in response to mechanical stimuli, and are hypothesized to be the functional linker between the extracellular matrix to the intracellular cytoskeleton [45]. The application of magnetic fields can be used to modulate the orientation and direction of neurite formation in cultured human neuronal cells [46]. In presence of a static magnetic field, neurites grow in a specific direction due to the application of magnetic fields change the co-localization of microtubule and actin filament in culture cells, that is, the subcellular polarity of the microtubule-actin interaction is induced by an external magnetic field [46]. Introduction 10 There is a force F generated on the attached MNPs when a magnetic particle with magnetic moment is placed in a non-uniform magnetic field. The force F creates a mechanical tension on the axon which stimulates nerve regeneration in the direction imposed by the magnetic field. Thus, magnetic nanoparticles would influence or stimulate the focal adhesion of the growth cone in the axon by mechanical stretch, in the direction imposed by the external magnetic field, and would act as a guidance cue to direct the neurite outgrowth (Figure 3). Figure 3. Nerve regeneration mediated by MNPs. MNPs bind the injured nerve, a magnetic field is thus applied in the direction of the nerve regeneration. MNPs create a mechanical tension which stimulates nerve regeneration in a direction imposed by the magnetic field. The physical guidance should direct more efficiently the regeneration of the injured nerve from the proximal to the distal stump. Additionally, MNP binding to the neuronal cells can be enhanced with biological molecules, e.g. neural binders and neurotrophic factors (NGF). The functionalization of MNPs with specific cellular ligands, able to bind selectively to the surface receptors of the neurons, could increase the specificity as well as the efficiency of the process. Objetives 11 II. OBJECTIVES The aim of this research project has been to investigate a novel approach to stimulate nerve regeneration based on the use of magnetic nanoparticles (MNPs), which can be manipulated by external magnetic fields. This ‘action at distance’, combined with the intrinsic penetrability of magnetic fields through human tissues, have been used to induce a mechanical force at neuron level in order to stimulate and initiate axon regrowth in the desired direction. In this work, I present a study that investigates the effects of iron oxide nanoparticles on the neurite outgrowth during differentiation in the presence of an external magnetic field. The present work had the following objectives: Didactic Objectives  To develop an experimental project with independence and originality.  To apply the theoretical knowledge to the interpretation and analysis of the experimental data.  To improve skill at oral and written communication, circulating the results and, interaction with colleagues and professionals from other disciplines. Experimental Objectives  Cell culture and measurement of magnetic nanoparticles uptake by a neural cell lineage (PC12).  To design experimental strategies handling biocompatible magnetic nanoparticles, cell cultures and magnetic fields, in the field of neural regeneration.  Characterization of PC12 cells after the treatment with magnetic nanoparticles by electronic microscopies (TEM and Dual Beam SEM-FIB analysis) and confocal optical microscopy. III. MATERIALS AND METHODS 1. Synthesis of PEI coated Fe PEI-Fe 3 O 4 nanoparticles. The PEIMNPs used in these experiments were synthesized described elsewhere [14] . Briefly, the magnetite oxidative hydrolysis method was dissolved in distilled water in a three solution containing FeSO 4 (previously flowed with N 2 precipitation a nitrogen flow was passed while temperature was maintained at 90º C for 24 h, and finally cooled to room temperature with an ice bath. The resulting black product was separated by magn to remove all the impurities and get physiological pH. Fluorescent labeling of PEI The free amine groups of PEI (Figure 4). 2.5 mg of PEI - bicarbonate buffer at pH 9.5 to ensure the deprotonation of amine groups on the PEI polymer coating. Alexa 488 solution (1 mg/ml DMSO: 20 PEI-MNPs susp ension and the reaction mixture suspension was covered foil and rotated (using a Rotator) at room temperature for 3 hours. The Alexa 488 labelled PEIMNPs were finally washed with deionized H fluorescence was observed in th Materials MATERIALS AND METHODS Synthesis of PEI coated Fe 3 O 4 nanoparticles (PEIMNPs) and fluorescent nanoparticles. MNPs used in these experiments were synthesized and characterized as . Briefly, the magnetite nanoparticles were synthesized by an oxidative hydrolysis method [7] . In a typical synthesis, a mixture of KNO was dissolved in distilled water in a three - necked flask bubbled with N 4 ·7 H 2 O and polyethyleneimine (PEI 25 kDa, 2 for 2 hrs) was added dropwise under constant stirring. After precipitation a nitrogen flow was passed while temperature was maintained at 90º C for 24 h, and finally cooled to room temperature with an ice bath. The resulting black product was separated by magn etic decantation and washed several times with Q to remove all the impurities and get physiological pH. Fluorescent labeling of PEI -Fe 3 O 4 nanoparticles (f-PEIMNPs) The free amine groups of PEI - MNPs were tagged with the fluorescent dye alexa 488 - MNPs were suspended in 1 ml of 0.1 M sodium carbonate bicarbonate buffer at pH 9.5 to ensure the deprotonation of amine groups on the PEI polymer coating. Alexa 488 solution (1 mg/ml DMSO: 20 μ l) was then added to the ension and the reaction mixture suspension was covered (using a Rotator) at room temperature for 3 hours. The Alexa 488 MNPs were finally washed with deionized H 2 O until no further fluorescence was observed in th e supernatant. The sample is denoted as f Materials and Methods 12 MNPs) and fluorescent and characterized as nanoparticles were synthesized by an . In a typical synthesis, a mixture of KNO 3 and NaOH necked flask bubbled with N 2 . A H 2 SO 4 (PEI 25 kDa, Figure. 4) 2 hrs) was added dropwise under constant stirring. After precipitation a nitrogen flow was passed while temperature was maintained at 90º C for 24 h, and finally cooled to room temperature with an ice bath. The resulting black etic decantation and washed several times with Q -water MNPs) MNPs were tagged with the fluorescent dye alexa 488 MNPs were suspended in 1 ml of 0.1 M sodium carbonate - bicarbonate buffer at pH 9.5 to ensure the deprotonation of amine groups on the PEI l) was then added to the ension and the reaction mixture suspension was covered with aluminum (using a Rotator) at room temperature for 3 hours. The Alexa 488 - O until no further e supernatant. The sample is denoted as f -PEI-MNPs. Materials and Methods 13 Figure 4. (a) Chemical structure of polyethyleneimine (PEI 25 kDa). (b) Alexa Fluor® 488 carboxylic acid, TFP ester, bis (triethylammonium salt); (MW ~885). 2. Physicochemical characterization of magnetic nanoparticles The obtained MNPs were characterized in terms of external morphology, microstructure, size distribution and magnetic properties. 2.1.Transmission electron microscopy (TEM) MNPs average size, distribution and morphology were analyzed by transmission electron microscopy (TEM) using a FEI Tecnai T20 microscope and operating at 200 keV. TEM samples were prepared by placing one drop of a dilute suspension of magnetite nanoparticles in water on a carbon-coated copper grid and allowing the solvent to evaporate at room temperature. The average particle size (DTEM) and distribution were evaluated by measuring the largest internal dimension of 200 particles. 2.2.Zeta potential The zeta potential and isoelectric point were evaluated at room temperature on a photo correlation spectrometer (PCS) Brookhaven 90 plus from a dilute suspension of the sample in water in a zeta-potential cuvette with 0.01 M concentration of KCl. 2.3.Magnetic characterization The magnetic characterization was carried out using a SQUID Magnetometer (Quantum Design MPMS-XL). Zero-field-cooled (ZFC) and field-cooled (FC) curves were measured between 5 K and 250 K, with cooling field H FC = 100 Oe. For the ZFC curves, the samples were first cooled from room temperature in zero applied field to the basal temperature (10 K). Then a field was applied and the variation of magnetization (M) was measured with increasing temperature up to T = 250 K. After the last point was measured, the FC curve was taken by cooling the sample keeping the same field; then the M vs. T data was measured increasing temperatures. Low-temperature hysteresis loops (10-250 K) were obtained in applied fields up to 5 T. Materials and Methods 14 3. In-vitro experiments 3.1.PC12 Cell culture Rat pheochromocytoma PC12 cells obtained from American Type Culture Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEN; Lonza, Verviers, Belgium) with 10 % horse serum, 5 % fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine in T-25 flasks (growth medium). Cells were maintained at 37 ºC in a saturated humidity atmosphere of 95 % air and 5 % CO 2 . Before cell seeding, well plates were coated with poly-l-lysine (PLL) (Sigma 81339). For cell differentiation, nerve growth factor (NGF, 80 ng/ml) was added into serum-reduced media (2% FBS). In-vitro experiments were designed at 10 μg/mL of PEI-MNPs. After the incubation time (24 h) the cells were washed and the modified-DMEM was replaced with ordinary DMEM. Control experiments were performed with growth medium without nanoparticles. When cultured for 5 days with or without magnets, the cell morphology and length and orientation of neurites were investigated. 3.2.Quantification of uploaded PEI-MNPs in PC12 cells Iron oxide nanoparticles content into PC12 cells was quantified by thiocyanate colorimetric assay. Cells were seeded (500,000 cells/mL) in 6-well dishes (1 mL growth medium and 500,000 cells/well) and treated with PEI-MNPs (5-50 µg/mL). After 24 hours of incubation, cells were isolated and lysed in HCl 6M-HNO 3 (65 %) for 2 h to dissolve all the components including nanoparticles. Samples were diluted with HCl 6 M and filtered. The iron content was determined by adding potassium thiocyanate to the Fe 3+ solutions in order to form the iron-thiocyanate complex, which has strong absorbance at 478 nm wavelength. The iron concentration was determined by comparing the sample absorbance to a calibration curve. Materials and Methods 15 3.3.SEM and Dual beam (FIB/SEM) analysis of PC12 cells incubated with PEI-MNPs To assess the cellular distribution of PEI-MNPs, scanning electron microscopy (SEM INSPECT F50, FEI Company) and dual-beam FIB/SEM (Nova 200 NanoLa, FEI Company) analysis images were taken in conditioned samples of PC12 neuron-like cells. SEM images were taken at 5 and 30 kV with a FEG column and a combined Gabased 30 kV (10 pA) ion beam was used to cross-sectioning single cells. These investigations were completed by energy-dispersive X-Ray spectroscopy (EDX) for chemical analysis. The preparation of the cells was made by seeding cells onto sterile glass cover slips (12 mm Ø) inside 35 mm dishes (Ibidi Petri dishes, cod. 80156, Ibidi, Germany) at a density of 25,000 cells/well in 800 μL of culture medium for 24 hours. After that, the growth medium was removed and replaced with medium containing 10 μg/mL MNPs. Then, the cells were induced to differentiate with NGF (80 ng/mL) and incubated for 4 days. After differentiation the cells were washed with PBS and fixed with 2 % glutaraldehyde solution for 2 hours at 4 ºC and washed again with PBS. Next, cells were incubated with 2 % osmium tetroxide and 2.5 % potassium ferrocyanide K 3 [Fe(CN) 6 ] for 1 hour at room temperature and darkness. Subsequently and after washing with 0.1 M phosphate-buffered saline (PBS), the cells were dehydrated via immersion in increasing concentrations of methanol 30 %, 50 %, 70 % and 90 % followed by further dehydration with anhydrous MeOH. After drying the samples were sputtered with 30 nm of gold. 3.4.Immunocytochemistry assay in PC12 cells: actin and tubulin fluorescent staining The preparation of cells for immunocytochemistry was made by seeding cells onto sterile 35 mm dishes (Ibidi Petri dishes, cod. 80156, Ibidi, Germany) previously treated with PLL, at concentration of 25,000 cells/well in 800 μL of culture medium and incubated overnight to allow cell adhesion. f-PEI-MNPs (Alexa Fluor® 488) were added at a concentration of 10 µg/mL and incubated 24 h, after that, cells were cultivated in differentiation medium for 4 days. The PC12 cells were fluorescent stained for tubulin and actin, separately. Materials and Methods 16 Phalloidin staining For actin staining, the PC12 cells were rinsed briefly with PBS and fixed with 3 % paraformaldehyde for 15 minutes. After washing, cells were blocked with 5 % bovine serum albumin in PBS for 15 min. After washing again with PBS, the cells were permeabilized by the addition of 1 % bovine serum albumin and 0.1 % saponin in PBS (permeabilization solution) and incubated for 1 hour. To visualize actin, samples were incubated with phalloidin-alexa fluoride® 546 (1:200) in BSA/saponin/PBS for 1 hour and finally, were rinsed again and put together in cover slips (20 mm Ø) with Mowiol/DAPI (1000:1). DAPI (4,6-diamidino-2-phenylindole 5 mg/mL) was used to visualize nuclei. All steps were performed at darkness and at room temperature. Tubulin staining The PC12 cells were rinsed with PBS and fixed with cold methanol for 15 min at -4 ºC. After washing, cells were blocked with 5 % bovine serum albumin in PBS for 15 min at room temperature. In this case, the permeabilization and the tubulin labeling were made simultaneously. After washing again with PBS, mouse monoclonal anti-neuronal class III beta tubulin (β-III-tubulin) primary antibody diluted 1:400 in permeabilization solution (BSA/saponin/PBS) was added to the samples in order to label tubulin and incubated for 1 hour at room temperature. Then, samples were rinsed in permeabilization solution, incubated for 1 h at room temperature with Alexa fluor® 633-conjugated goat anti-mouse secondary antibody diluted 1:500 in permeabilization buffer and lastly, assembled with Mowiol/DAPI in cover slips (20 mm Ø). Finally, all the assembled samples were dried and analyzed by confocal microscopy. 4. Neurites orientation and outgrowth: experimental set-up PC12 cells (800 cells/cm 2 ) were seeded in T-25 flasks previously treated with poly-Llysine coating (PLL, 10 µg/mL) which gives the cover glass an effective positive charge that increases cellular adhesion. After 24 hours, the cells were incubated with 10 µg/mL of PEIMNPs overnight. The next day, the medium-containing PEI-MNPs was replaced Results 23 10 µg/mL 20 µg/mL 50 µg/mL 100 µg/mL Cytotoxicity (%) 1.4 ± 0.4 1.3 ± 0.5 2.2 ± 0.4 3.8 ± 0.8 Table 1. Effect of PEI-MNPs on PC12 cell line. Cell viability was carried out by PI exclusion after 72 h of incubation. Cell counting was carried out using FACS analysis (mean ± s.e.m., n=6, p<0.0001). As a general result, the data on the viability of PC12 cells performed by Propidium Iodide exclusion assays (Table 1) displayed a toxicity level that increased in concentration-dependent manner. The toxicity was less at short incubation times but little important effects were noticed for incubation times longer than 48 h and large MNPs concentrations (Table 1). In spite of the effect for the largest MNPs concentrations, it is worth to note that these values correspond to incubation conditions (e.g., 72 hours and 100 µg/ml) that largely exceed therapeutic values. 2.2. Quantification of uploaded PEI-MNPs in neural cells The intracellular iron content for the PEI coated MNPs in PC12 cell line is shown in Figure 10. After incubating cells with various concentrations of PEI-MNPs (from 5 µg/mL to 50 µg/mL of MNPs) in PC12 cells, we observed that when we increased the amount of PEI-MNPs incubated with the cells, the intracellular uptake of our positively charged MNPs also increased. Thus, significant nanoparticle internalization was observed for PEI-MNPs after 24 hours of nanoparticle incubation in the culture medium. It is possible that the PEI-MNP attachment to the membrane and the PEI-MNP internalization was facilitated by their positively charged surface. At the working concentration (10 µg/mL of PEI-MNPs), PC12 cells incorporated around 10 pg Fe per cell; this value includes both MNPs attached to the cell membrane by electrostatic attraction and MNPs inside the cell. Figure 10. Total amount of iron content per cell at different concentration of 2.3. Biodistribution of PEI The cellular localization of particles after incubating with PEI of 10 µg/m L was investigated using TEM analysis ( 0 5 10 15 20 25 30 35 40 Control (0) Amount of Fe (pg Fe/cell) Total amount of iron content per cell at different concentration of PEI-MNPs added. Biodistribution of PEI -coated MNPs in neuronal cells The cellular localization of particles after incubating with PEI - MNPs at a concentration L was investigated using TEM analysis ( Figure 11) [14]. 5 µg/mL 10 µg/mL 20 µg/mL 50 µg/mL Concentration of PEI-MNPs Results 24 Total amount of iron content per cell at different concentration of MNPs at a concentration 50 µg/mL Figure 11. Cellular localization of PEI h with f-PEI-MNPs 10 µg /mL. particles entering and membrane invagination; c cells incub ated 24 h with PEI spectra of citoplasmatic PEI inside the cell. TEM images revealed the presence of cluster of particles inside cells ( white arrows) and on the cell membrane (Figure. 11 were detected in the nucleus. The presence of Fe inside the cells was confirmed by EDS– HAADF spectra (Figure 11 c The cellular localization of particles after of 10 µg/mL was investigated using SEM and Dual Beam analysis. The PEI biodistribution was investigated after incubation in two conditions: cell cultures with PEIMNPs for 24 h, differentiated by NGF induct field (named MNP + H - ); the other condition was cell cultured with MNPs for 24 h, differentiated by NGF induction and differentiated in the presence of an external magnetic field (named MNP SEM images showed that afte attached to the soma and growth cone in both, control cells (without magnetic field) and cells subjected to magnetic field, forming large clusters ranging from 1 to 5 µm in Figure 11. Cellular localization of PEI -MNPs. ab) TEM analysis of PC12 cells incubated 24 /mL. White arrows: particles in the cytoplasm, yellow arrows: particles entering and membrane invagination; c -d) STEMHAADF images (100 K) of PC12 ated 24 h with PEI -MNPs 10 µg/mL. The inset of panel d) shows the spectra of citoplasmatic PEI -MNPs. The EDSHAADF spectra confirm the presence of Fe TEM images revealed the presence of cluster of particles inside cells ( white arrows) and on the cell membrane (Figure. 11 a-b , yellow arrows). No particles were detected in the nucleus. The presence of Fe inside the cells was confirmed by HAADF spectra (Figure 11 c -d). The cellular localization of particles after incubating with PEIMNPs at a concentration of 10 µg/mL was investigated using SEM and Dual Beam analysis. The PEI biodistribution was investigated after incubation in two conditions: cell cultures with MNPs for 24 h, differentiated by NGF induct ion and without external magnetic ); the other condition was cell cultured with MNPs for 24 h, differentiated by NGF induction and differentiated in the presence of an external magnetic field (named MNP + H + ). SEM images showed that afte r incubation, a fraction of PEIMNPs were strongly attached to the soma and growth cone in both, control cells (without magnetic field) and cells subjected to magnetic field, forming large clusters ranging from 1 to 5 µm in Results 25 b) TEM analysis of PC12 cells incubated 24 White arrows: particles in the cytoplasm, yellow arrows: HAADF images (100 K) of PC12 The inset of panel d) shows the EDS-HAADF HAADF spectra confirm the presence of Fe TEM images revealed the presence of cluster of particles inside cells ( Figure 11 a-b, , yellow arrows). No particles were detected in the nucleus. The presence of Fe inside the cells was confirmed by MNPs at a concentration of 10 µg/mL was investigated using SEM and Dual Beam analysis. The PEI -MNPs biodistribution was investigated after incubation in two conditions: cell cultures with ion and without external magnetic ); the other condition was cell cultured with MNPs for 24 h, differentiated by NGF induction and differentiated in the presence of an external MNPs were strongly attached to the soma and growth cone in both, control cells (without magnetic field) and cells subjected to magnetic field, forming large clusters ranging from 1 to 5 µm in length. In agreement with SEM im were PEIMNPs and were strongly attached to the cell membrane (Figure 12). Red color gives us an idea of the amount of iron over the cell. Figure 12. Scanning electron microscopy image of a single PC1 with 10 µg/mL PEIMNPs, showing the presence of PEI on the cell membrane and on the growth cone. analysis showing Fe in red and O in green. (b) PC12 cells subjected to magnet length. In agreement with SEM im ages, EDX analysis confirmed that these bright spots MNPs and were strongly attached to the cell membrane (Figure 12). Red color gives us an idea of the amount of iron over the cell. Figure 12. Scanning electron microscopy image of a single PC1 2 cell incubated for 24 hours MNPs, showing the presence of PEI - MNPs agglomerates (bright spots) on the cell membrane and on the growth cone. (a) PC12 cells without magnetic field and EDX analysis showing Fe in red and O in green. (b) PC12 cells subjected to magnet Results 26 ages, EDX analysis confirmed that these bright spots MNPs and were strongly attached to the cell membrane (Figure 12). Red 2 cell incubated for 24 hours MNPs agglomerates (bright spots) (a) PC12 cells without magnetic field and EDX analysis showing Fe in red and O in green. (b) PC12 cells subjected to magnet ic field. The combination of Ion and Electron beam (SEM/FIB) allows the study of the internal distribution of particles in adherent cells because it allows us to see the PEI inside the crosssectioned cells. For further study of the internalization of sectioned by FIB and analyzed by SEM after culture in the presence of 10 PEIMNPs for 24 hours. The results were different for PC12 cells cultured in the presence of an external magnetic field (MNP magnetic field (MNP + H - ). In the Figure 13 it is possible to observe the agglomeration of the PEIMNPs attached to the outside part of the cell membrane in MNP condition, and also the agglomerates c neural soma. It is important to point out that we did not find MNPs in the intracellular space of the axon, we only found MNPs in the intracellular part of the neural soma of MNP + H - cells. Figure 13. SEM ima ges of PC12 cells (MNP incubation at 10 µg/mL of PEI 52 º tilted previously to be cross we could observe the absence of PEI could see the presence of PEI space. The combination of Ion and Electron beam (SEM/FIB) allows the study of the internal distribution of particles in adherent cells because it allows us to see the PEI sectioned cells. For further study of the internalization of the PEIMNPs, single PC12 cells were cross sectioned by FIB and analyzed by SEM after culture in the presence of 10 MNPs for 24 hours. The results were different for PC12 cells cultured in the presence of an external magnetic field (MNP + H + ) and PC12 cells cultured without ). In the Figure 13 it is possible to observe the agglomeration MNPs attached to the outside part of the cell membrane in MNP condition, and also the agglomerates c ould be seen along the intracellular space of the neural soma. It is important to point out that we did not find MNPs in the intracellular space of the axon, we only found MNPs in the intracellular part of the neural soma of ges of PC12 cells (MNP + H - ) crosssectioned by FIB after 24 hours incubation at 10 µg/mL of PEI -MNPs and for 4 days of differentiation. (a) The whole cell at 52 º tilted previously to be cross -sectioned. (b) And (c) i ntracellular space of the axon in where we could observe the absence of PEI - MNPs. (d) Neural soma intracellular space in where we see the presence of PEI - MNPs in both attached to the membrane and in the intracellular Results 27 The combination of Ion and Electron beam (SEM/FIB) allows the study of the internal distribution of particles in adherent cells because it allows us to see the PEI -MNPs MNPs, single PC12 cells were cross - sectioned by FIB and analyzed by SEM after culture in the presence of 10 μg/mL of MNPs for 24 hours. The results were different for PC12 cells cultured in the and PC12 cells cultured without ). In the Figure 13 it is possible to observe the agglomeration MNPs attached to the outside part of the cell membrane in MNP + H - be seen along the intracellular space of the neural soma. It is important to point out that we did not find MNPs in the intracellular space of the axon, we only found MNPs in the intracellular part of the neural soma of sectioned by FIB after 24 hours (a) The whole cell at ntracellular space of the axon in where MNPs. (d) Neural soma intracellular space in where we MNPs in both attached to the membrane and in the intracellular As we can see in the Figure 14 we could find agglomeration of the MNPs in the intracellular part along th to occupy a major fraction of the intracellular space. EDX spectra show agglomerates were composed of iron. The MNPs distribution observed consist MNPs agglomerates attached to the cell membrane, which seem also to penetrate to the intracellular space of the soma and the axon. Figure 14. SEM images of PC12 cells (MNP incubation at 10 µg/mL of PEI 52 º tilted previously to be cross could observe the presence of PEI inside the axon. (c ) Neural soma intracellular space in where we could see the presence of PEI MNPs. The insets c orrespond with the EDX spectra of the highlight part. The microscopy imaging analysis of the PEI kinetics of particle uptake could go through progressive stages like a) a first attachment to the cell membrane as cle crossing reflecting early endocytosis (with pathways not yet known), and accumulation in membrane through which MNP uptake is media The results from SEM and Dual Beam microscopy ( incubation the final distribution at the cellular level consisted of large As we can see in the Figure 14 (see also Figure A1) , in the case of MNP we could find agglomeration of the MNPs in the intracellular part along th to occupy a major fraction of the intracellular space. EDX spectra show composed of iron. The MNPs distribution observed consist MNPs agglomerates attached to the cell membrane, which seem also to penetrate to the intracellular space of the soma and the axon. Figure 14. SEM images of PC12 cells (MNP + H + ) crosssectioned by FIB after 24 hours incubation at 10 µg/mL of PEI -MNPs and for 4 days of differentiation. (a) The whole cell at 52 º tilted previously to be cross -sectioned. (b ) Intracellular space of the axon in where we could observe the presence of PEI - MNPs agglomerates in both attach to the membrane and ) Neural soma intracellular space in where we could see the presence of PEI orrespond with the EDX spectra of the highlight part. The microscopy imaging analysis of the PEI -MNPloaded PC12 cells indicated that the kinetics of particle uptake could go through progressive stages like a) a first attachment to the cell membrane as cle arly observed from SEM images; b) a subsequent membrane crossing reflecting early endocytosis (with pathways not yet known), and accumulation in membrane - bound intracellular vesicles. The detailed mechanisms through which MNP uptake is media ted by PEI is still to be determined. The results from SEM and Dual Beam microscopy ( Figure 13 and 14) showed that, after incubation the final distribution at the cellular level consisted of large Results 28 , in the case of MNP + H + condition, we could find agglomeration of the MNPs in the intracellular part along th e whole cell to occupy a major fraction of the intracellular space. EDX spectra show ed that these composed of iron. The MNPs distribution observed consist ed of MNPs agglomerates attached to the cell membrane, which seem also to penetrate to the sectioned by FIB after 24 hours (a) The whole cell at ) Intracellular space of the axon in where we MNPs agglomerates in both attach to the membrane and ) Neural soma intracellular space in where we could see the presence of PEI - loaded PC12 cells indicated that the kinetics of particle uptake could go through progressive stages like a) a first attachment arly observed from SEM images; b) a subsequent membrane crossing reflecting early endocytosis (with pathways not yet known), and c) progressive bound intracellular vesicles. The detailed mechanisms ted by PEI is still to be determined. 13 and 14) showed that, after agglomerates of Results 29 PEI-MNPs both attached to the cell membrane and at the intracellular space. The capacity provided by the Dual Beam technique, of analyzing the biodistribution of the MNPs at the single-cell level, demonstrated also that the agglomerates observed outside the cellular membrane extended in a continuous way into the cytoplasm. The results also showed that the magnetic field promote the retention and accumulation of PEIMNPs in cells and can facilitate the movement of these PEI-MNPs from the soma to the axon growth cone or their incorporation in the axon. The combination of PEI-MNPs and an external magnetic field can provide mechanical tensile forces in the growth cone that may cause axonal sprouting [49]. Since the PEI-MNPs did not show major toxicity effects except for the largest concentrations and after long incubation time (72 h) [14], the final distribution of large magnetic agglomerates anchored to the PC12 cells opens the possibility of using external fields for cellular actuation through external magnetic forces on these structures. These results also confirmed that there were no particles in the nucleus. 2.4. Immunofluorescence analysis of neuronal cell cytoskeleton Morphological changes in the PC12 cells during differentiation were also affected by the PEI-MNPs (Figure 15 and Annex section, Figure A2). These differences could be observed and analyzed through phase contrast and immunofluorescence microscopy. Typical PC12 cells, prior to NGF exposure, are undifferentiated, spherical in shape and do not produce neurites, as can be seen in Figure 15a. Following exposure to NGF, as shown in Figure 15b, PC12 cells differentiate into neuronal type cells (+NGF) and begin to extend short and thin neurites (5-6 per cell) into the periphery. PC12 cells exposed to PEI-MNPs were more elongated, showed less number of neurites (around 23 per cell), and these were longer and sharper (Figure 15c). The number of connections or neuronal synapses also increased in the presence of MNPs. Figure 15. Phase contrast images of PC12 cells: MNPs - ), (c) with NGF and PEI the 4 th day of inducing differentiation. These results indicate that PEI outgrowth relative to nontreated cells. PC12 cells incubated with PEI field exhibited neurites outgrowth in the 16 and Annex section, Figure A3 but differentiated in the presence of the magnetic field display neurites and randomly oriented (Figure Phase contrast images of PC12 cells: (a) undifferentiated, (b) with NGF (NGF ), (c) with NGF and PEI -MNPs (NGF + , PEI-MNPs + ). The b and c images were recorded day of inducing differentiation. These results indicate that PEI - MNPs promoted neurite treated cells. PC12 cells incubated with PEI - MNPs and differentiated in the presence of the magnetic neurites outgrowth in the direction of the applied magnetic field (Figure and Annex section, Figure A3 ). Neurites of PC12 cells did not treat with PEI but differentiated in the presence of the magnetic field display ed higher number of neurites and randomly oriented (Figure s 15 and 16). Results 30 (a) undifferentiated, (b) with NGF (NGF + , ). The b and c images were recorded MNPs promoted neurite MNPs and differentiated in the presence of the magnetic direction of the applied magnetic field (Figure not treat with PEI -MNPs higher number of Figure 16. Phase contrast images of PC12 cells differentiated in the presence of a magnetic field (MNP of a magnetic field (MNP - H + magnetic field (MNP + H - ). The changes in morphology and cytoskeletal structure of cells exposed to PEI and a magnetic field (H) were also investigated using immuno and Factin. The cells were fixed, permeabilized and stained with fluorescently labeled secondary antibody to primary anti microfilaments were labeled with phalloidin (Figure 17 section). Untreated cells (MNP - ) exhibited strong peripheral F indicative of cortical actin fibers. Also, the actin microfilaments were well organized on the top of neurites forming filopodia Phase contrast images of PC12 cells : (a) incubated with PEI differentiated in the presence of a magnetic field (MNP + H + ), (b) differentiated in the presence + ) and, (c) incubated with PEIMNPs in the absence of an external The changes in morphology and cytoskeletal structure of cells exposed to PEI and a magnetic field (H) were also investigated using immuno fluorescence for actin. The cells were fixed, permeabilized and stained with fluorescently labeled secondary antibody to primary anti -tubulin antibodylabeled microtubules, while actin microfilaments were labeled with phalloidin (Figure 17 and Figures A4 and A5, Annex ) exhibited strong peripheral F - actin staining along the cell edge, indicative of cortical actin fibers. Also, the actin microfilaments were well organized on the top of neurites forming filopodia and lamellipodia (Figure 17). Results 31 (a) incubated with PEI -MNPs and ), (b) differentiated in the presence MNPs in the absence of an external The changes in morphology and cytoskeletal structure of cells exposed to PEI -MNPs fluorescence for β tubulin actin. The cells were fixed, permeabilized and stained with fluorescently labeled labeled microtubules, while actin and Figures A4 and A5, Annex actin staining along the cell edge, indicative of cortical actin fibers. Also, the actin microfilaments were well organized on At the same day of differentiation (4 unpolarized shape and, had short undifferentiated neurites (Figure microtubules also form a dense network equally distributed around along the neurites of the cells (Figure 18). In the case of cells incubated with PEI (MNP + ), there were prominent filopodia and lamellipodia on the top of neurites; the cells were polarized and possessed two types of morphologically and functionally distinct processes; a single, long, and slender axon and several dendrites that are shorter processes with a tapered morphology ( Figure 17. Immunofluorescence images of d after treatment with f-PEIMNPs (b). Red fluorescence represents actin microfilaments and green color represents fluorescent treatment; the inset of (a) is a detail of PC12 cell that exhibited spherical unpolarized shaped At the same day of differentiation (4 th day) untreated cells exhibited spherical unpolarized shape and, had short undifferentiated neurites (Figure microtubules also form a dense network equally distributed around along the neurites of the cells (Figure 18). In the case of cells incubated with PEI ), there were prominent filopodia and lamellipodia on the top of neurites; the polarized and possessed two types of morphologically and functionally distinct processes; a single, long, and slender axon and several dendrites that are shorter processes with a tapered morphology ( Figure 17b). Figure 17. Immunofluorescence images of d ifferentiated PC12 cells 4 days MNPs (b). Red fluorescence represents actin microfilaments and fluorescent nanoparticles . (a) PC12 cells without treatment; the inset of (a) is a detail of PC12 cell that exhibited spherical unpolarized shaped Results 32 day) untreated cells exhibited spherical unpolarized shape and, had short undifferentiated neurites (Figure 17a). The microtubules also form a dense network equally distributed around the nucleus and along the neurites of the cells (Figure 18). In the case of cells incubated with PEI -MNPs ), there were prominent filopodia and lamellipodia on the top of neurites; the polarized and possessed two types of morphologically and functionally distinct processes; a single, long, and slender axon and several dendrites that are shorter ifferentiated PC12 cells 4 days control (a) and MNPs (b). Red fluorescence represents actin microfilaments and . (a) PC12 cells without f-PEI-MNPs treatment; the inset of (a) is a detail of PC12 cell that exhibited spherical unpolarized shaped Discussion 39 V. DISCUSSION The work reported here has been conceived within the wider framework of a research project aimed to validate a novel protocol for nerve regeneration based on the use of magnetic nanoparticles and external static magnetic fields. The hypothesis on which this research project was based is schematically depicted in Figure 23. When an injury occurs to a peripheral nerve, magnetic nanoparticles can be used to guide the regenerating axons across the traumatic gap by exposure to a magnetic field which exerts a magnetic force in the direction of the regenerating axon (from the proximal end to the distal portion). MNPs create a mechanical tension which stimulates nerve regeneration in the direction imposed by the magnetic field. This physical guidance should direct more efficiently the regeneration of the injured nerve from the proximal toward the distal stump. Additionally, MNP binding to the neuronal cells can be enhanced with addition of biological molecules, e.g., neural binders and neurotrophic factors (NF). Figure 23. Nerve regeneration mediated by MNPs. After a nerve transaction, Schwann cells, macrophages and monocytes work together to remove myelin debris, release neurotrophins, and lead axons toward their synaptic targets. MNPs bind the injured nerve, a magnetic field is thus applied in the direction of the nerve regeneration. MNPs create a mechanical tension which stimulates nerve regeneration in a direction imposed by the magnetic field. This physical guidance should direct more efficiently the regeneration of the injured nerve from the proximal toward the distal stump. Discussion 40 Additionally, MNP binding to the neuronal cells can be enhanced with biological molecules, e.g., neural binders and neurotrophic factors (NF). Although many types of magnetic nanoparticles are currently used in clinical protocols, the use of iron oxides nanoparticles in neurosciences is still limited due to the need of clear data on possible toxicity effects, the effects of poor water solubility and the nanometric size. Therefore the potential adverse effects of iron oxide nanoparticles are currently the focus of intense investigation [50], and measures to reduce adverse effects still remain explored [51]. Within this context, the present work was aimed to constitute a proof of principle for the magnetically-driven neurite orientation. Specifically, neuronal PC12 cells loaded with PEI-coated magnetic nanoparticles (PEI-MNPs) were used in order to demonstrate that a static magnetic field can orientate the orientation of neurites outgrowth in an in vitro model. A first issue to note from the systematic analysis of different microscopic techniques (TEM, SEM, Dual-beam, confocal, fluorescence) is that there is a strong interaction between PEI-MNPs and PC12 cells. The results obtained from SEM and Dual Beam microscopy (See Figures 12, 13 and 14 of Results section-) showed that after incubation with MNPs the final distribution at the cellular level consisted of large agglomerates of MNPs both attached to the cell membrane and at the intracellular space. Indeed, the agglomeration of MNPs in biological media is an unavoidable process since the large concentration of proteins in cell culture media inevitably led to the formation of a protein ‘corona’ and, more generally, the formation of large aggregates. It is this kind of aggregates that constitute the entities to interact with cells and tissues in biological applications. The analysis of the biodistribution of MNPs at the single-cell level through Dual Beam technique demonstrated that the agglomerates observed outside the cellular membrane extended in a continuous way into the cytoplasm. Since the MNPs resulted in a very low toxicity effects at the concentrations used for these experiments, the final distribution of large magnetic agglomerates anchored to the PC12 cells seems to be potentially useful for cellular actuation through external magnetic forces. The Discussion 41 internalization of these particles was also demonstrated by confocal microscopy. Interestingly, after incubation of the cells with PEI-MNPs and differentiation in the presence of an external magnetic field, we observed an enhanced presence of MNPs localized at the intracellular part of neural soma and axon. The experimental results confirmed that the synergic application of MNPs and magnetic field gradient influence the orientation of the neurite growth. When cells were treated with the PEI-MNPs and exposed to the external magnetic field, the neurites tended to grow parallel to the field direction (see Figures 19, 20 and 21 of Results section 3). The experimental controls demonstrated that the magnetic field alone did not influence the direction of the neurites growth. An influence of static magnetic fields onto the direction of newly formed neuronal processes of PC12 cells was reported by Kim et al. [46]. Specifically, the authors found that neurite grew preferentially in a direction perpendicular to the direction of the magnetic field. Unfortunately, the results cannot be compared because the experimental set-ups are different. Here the configuration of the magnets were designed in order to have a maximum magnetic field gradient (0.019 T/m) in the culture dish, i.e., all cells were exposed to the same magnetic force which is proportional to the magnetic field gradient and independent from the magnetic field value. On the opposite, Kim and colleagues used 12-well plate place between two permanent magnets with opposing poles facing each other to generate a magnetic field gradient (0,12 T at the center) and thus a magnetic force not constant. The morphological changes in the PC 12 cells observed during differentiation and when PEI-MNPs were added to the cell culture were already mentioned in Section 3.2 of Results. It is clear from the analysis of the neurite length distributions that a measurable increase in the average neurite length was induced on the PEI-MNPs-loaded cells when compared to the different control samples (i.e., blank cells with no MNPs, with and without applied external magnetic field). The differences of average neurite lengths between control and MNP-loaded samples (see Figure 22 of the Results section 3) was found to be about 10 µm (control length: 23-24 µm; MNP-loaded samples length: 32-33 µm). These differences were observed irrespective of the presence of magnetic field, indicating that its origin is related to the incorporation of MNPs. This observation (that Discussion 42 the presence of the PEI-MNPs during cell differentiation improves the outgrowth of the neurites) suggests that the PEI-MNPs could have positive effects on neurite growth, but determining the exact source of this enhancing neurite factor would need further investigation. This is consistent with a previous work by Kim et al. [37] reported that MNPs alone (i.e., in the absence of the magnetic field) was able to enhance the neurite outgrowth in a dose-dependent manner on PC12 cells exposed to both MNPs and NGF. They concluded that treatment of PC12 cells with iron ion promotes neurite outgrowth during NGF-induced differentiation by enhancing cell attachment and survival via activation of cell adhesion molecules. Indeed, it has been reported [52, 53] that Fe ions can affect the neural plasticity and neurite outgrowth, but the molecular mechanisms of this influence has not been described in detail. Some groups have reported that the treatment of PC 12 cells with iron ion promoted neurite outgrowth during NGF-induced differentiation by enhancing cell attachment and survival [52, 53]. Since, it is possible that iron oxide nanoparticles can be ionized due to the acidic pH inside the endosome or lysosome and the Fe ions can be released into the cell cytosol. These studies concluded that iron oxide nanoparticles might enhance neurite outgrowth by increasing the intracellular concentration of Fe ions during the differentiation process [52, 53]. However, it is still possible that the iron oxide nanoparticles activate the extracellular receptor that in turn may induce the subsequent signal transduction for neurite outgrowth [37]. Further studies are necessary to identify the key factors that affect the differentiation pathway by iron oxide nanoparticles. In spite of the increased value of the average neurite length observed for MNP-loaded PC12 cells, the average number of neurites <N N > counted for the same PC12 cells incubated with PEI-MNPs was smaller than the corresponding average neurite number in control cells.. The diminution of neurite formation after treatment with MNPs was also observed by Pisanic and co-workers [54]. They reported that the presence of intracellular anionic-MNPs appears to affect cytoskeletal structure by either chemically or physically impeding actin microfilament formation in the cells or inhibiting the maturation and function of neurites. It is clear from the experimental results that the driving force guiding the neurite growth along the magnetic field direction is the force exerted on the incorporated magnetic nanoparticles by the external magnetic field. In other words, the preferred orientation is Discussion 43 based on the interaction between the magnetic moment of the MNPs and the external magnetic field. However, the detailed biochemical mechanisms by which these forces acting on the MNPs translate into a preferential growth direction for a large fraction of the neurites is not yet fully understood. Some models have been proposed to simulate how a mechanical stimulation can activate cellular signaling pathways that are known as mechanotransduction pathways [41]. The process by which cells convert physical force into a biochemical signal plays a key role in the outgrowth and migration through its influence over cell function. The predominant molecular transducers of force are integrins, which are a family of glycoproteins that act as extracellular matrix receptors. Integrins play an essential role in the signaling and structure of a cell and interacts with the cytoskeleton through focal adhesion proteins, which regulate cell survival, differentiation, migration, and mechanotransduction pathways [45]. Kim et al. [37] demonstrated that magnetic nanoparticles incremented the expression of β1-integrin which might led to neurite outgrowth and are involved in cellular response to mechanical forces. In addition, magnetic beads coated with β1-integrin ligand showed force-dependent focal adhesion formation, indicating that integrins can transfer external force through the plasma membrane [41]. We have demonstrated that PEI-MNPs can both penetrate inside PC12 cells through an endocytic-like mechanism and also remain attached to the cell membrane. These results led us to conclude that the uptake of PEI-MNPs by neural cells may increment the expression of integrins that favor the formation of focal adhesion proteins in the direction of the applied magnetic field resulting in the formation of neurites in this direction. To sum up, our results have shown that PC12 neural cells with incorporated PEI-MNPs can preferentially direct the growth of neurites along the direction of an externally applied magnetic field. Moreover, in this process both the cell morphology and the number of the neurites are modified. The mechanism underlaying this neurite orientation has been demonstrated to depend on the magnetic forces acting on the MNPbound neurites. Our findings have established the proof of concept that MNPs can be employed as enhancers of nerve regeneration under the effect of externally applied Discussion 44 magnetic fields. This could be the first stage for a non-invasive strategy of neural regeneration based on nanotechnological devices. Conclusions and future perspectives 45 VI. CONCLUSIONS AND FUTURE PERSPECTIVES Nerve regeneration is a major issue in the treatment of traumatic nerve injuries and has stimulated considerable bioengineering research aimed at exploring innovative strategies for physical or chemical guidance of axonal re-growth across the nerve lesion. The project outlined in this work proposes a novel minimally invasive approach for physical guidance based on synergistic use of magnetic nanoparticles (MNPs) and magnetic fields (H). In this study, we attempted to determine whether the application of a magnetic field can affect neuron-like cells (PC12 cell line) previously incubated with polyethyleneiminecoated nanoparticles (PEI-MNPs). The selected PEI-MNPs were suitable for this purpose because they possess high saturation magnetization, negligible cytotoxicity and high capability to magnetize mammalian cells. The intracellular distribution of the PEIMNPs analyzed at the single-cell level by dual-beam (FIB/SEM) technique revealed the coexistence of clusters of PEI-MNPs attached to the membrane and crossing the cell membrane as well as in the intracellular part of the PC12 cells. MNPs were used to generate tensile forces under the effect of an external magnetic field and to manipulate axons in order to stimulate neurite initiation or axon elongation in the desired direction. When PC12 cells were cultured with the PEI-MNPs and exposed to an external magnetic field, particles were found in the cell body but also in the growth cone of developing neurites. In this neuron-like cell line, it was confirmed that PEIMNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field. The PEI-MNPs also affected the neurite length and cell morphology. Longer average of neurite length was found in cells loaded with PEI-MNPs. In summary, these results showed that the use of MNPs and magnetic forces seems to be a good and novel strategy for neural manipulation. References 46 VII. REFERENCES 1. Jain, K.K., Advances in the field of nanooncology. Bmc Medicines, 2010. 8. 2. Gupta, A.K.a.G., M., Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials, 2005. 26(18): p. 3995-4012. 3. 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Immunofluorescence images of differentiated PC12 cells 4 days after treatment with f-PEIMNPs in the presence of magnetic field (MNP fluorescence represents tubulin microtubules and green color represents nanoparticle reflection and yellow line represents the m PC12 cells stained with anti axon of the PC12 cells in which it is observed the PEI Immunofluorescence images of differentiated PC12 cells 4 days after MNPs in the presence of magnetic field (MNP fluorescence represents tubulin microtubules and green color represents nanoparticle reflection and yellow line represents the m agnetic field direction. (a) And (b) MNP PC12 cells stained with anti - tubulin III antibodies. The inset images represent the detail of axon of the PC12 cells in which it is observed the PEI - MNPs inside the axon. Annex 55 Immunofluorescence images of differentiated PC12 cells 4 days after MNPs in the presence of magnetic field (MNP + H + ). Red fluorescence represents tubulin microtubules and green color represents nanoparticle agnetic field direction. (a) And (b) MNP + H + tubulin III antibodies. The inset images represent the detail of MNPs inside the axon.