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Magnetic Hyperthermia in Dendritic Cells and Magnetofection in Brain Cells

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Magnetic Hyperthermia in Dendritic Cells and Magnetofection in Brain Cells Tesis Doctoral BIOMEDICAL APPLICATIONS OF MAGNETIC NANOPARTICLES: MAGNETIC HYPERTHERMIA IN DENDRITIC CELLS AND MAGNETOFECTION IN BRAIN CELLS Autor Asín Pardo, Laura Director/es Ibarra García, Manuel Ricardo Goya Rossetti, Gerardo Fabián FACULTAD DE CIENCIAS Departamento de Física de la Materia Condensada 2012 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es BIOMEDICAL APPLICATIONS OF MAGNETIC NANOPARTICLES: MAGNETIC HYPERTHERMIA IN DENDRITIC CELLS AND MAGNETOFECTION IN BRAIN CELLS. Memoria presentada para optar al grado de Doctor Europeo por la Universidad de Zaragoza Laura Asín Pardo Directores: M. Ricardo Ibarra Gerardo F. Goya Departamento de Física de la Materia Condensada Zaragoza, Marzo 2012. 1 Index Index .......................................................................................................................... 1 Abbreviations ............................................................................................................ 5 Preface ....................................................................................................................... 8 I. Introduction .................................................................................................... 11 I.I. Nanoscience and nanoparticles ............................................................. 13 I.II. Magnetism basis ................................................................................... 17 I.III. Magnetic hyperthermia ......................................................................... 26 I.IV. Dendritic cells ....................................................................................... 35 I.V. Magnetofection ..................................................................................... 47 II. Materials & Methods ...................................................................................... 53 II.I. AMF experiments in dendritic cells ..................................................... 55 II.I.I. Magnetic nanoparticles .................................................................. 56 II.I.II. Dendritic cells culture and MNPs loading ................................... 59 II.I.III. Viability assay ............................................................................. 62 II.I.IV. Maturation markers expression measurement by FACS ............ 67 II.I.V. Cytokines expression analysis ..................................................... 68 II.I.VI. Quantification of MNPs loaded per cell ..................................... 70 II.I.VII. Internalization study by FACS .................................................. 72 II.I.VIII. AMF application experiments .................................................. 72 II.I.IX. Scanning electron microscopy .................................................... 75 II.I.X. Transmission electron microscopy............................................... 76 II.I.XI. Dual Beam .................................................................................. 77 II.I.XII. Confocal microscopy ................................................................. 78 II.I.XIII. Western-Blot ............................................................................ 80 2 II.II. Preparation of Adenovirus-MNPs complexes for magnetofection ........ 83 II.II.I. Cell lines ....................................................................................... 84 II.II.II. Magnetic nanoparticles ............................................................... 85 II.II.III. Adenovirus titration ................................................................... 87 II.II.IV. Adenovirus-MNPs complexes association and stability study.. 88 II.II.V. Size, Z-potential and magnetic responsiveness measurements for complexes ............................................................................................... 90 II.II.VI. AFM .......................................................................................... 91 II.II.VII. TEM ......................................................................................... 92 II.II.VIII. Complexes internalization ...................................................... 92 II.II.IX. Viability assay ........................................................................... 94 II.II.X. Infection experiments ................................................................. 94 III. Results & Discussion ...................................................................................... 97 III-I: DCs and MNPs interaction .................................................................... 99 III.I.I. Characterization of the magnetic nanoparticles ......................... 101 III.I.II. DCs differentiation from PBMCs ............................................. 109 III.I.III. Viability assay ......................................................................... 111 III.I.IV. TEM......................................................................................... 117 III.I.V. SEM .......................................................................................... 120 III.I.VI. Dual Beam ............................................................................... 124 III.I.VII. Confocal microscopy ............................................................. 126 III.I.VIII. Quantification ....................................................................... 131 III.I.IX. Internalization study by FACS ................................................ 138 III.I.X. Membrane molecules expression profile .................................. 140 III.I.XI. Cytokine expression ................................................................ 147 III.I.XII. Conclusions ............................................................................ 150 III-II: Magnetic Fluid Hyperthermia in DCs ................................................ 151 III.II.I. AMF effect ............................................................................... 153 3 III.II.II. HSP-70 expression .................................................................. 167 III.II.III. SEM ........................................................................................ 169 III.II.IV. Conclusions ............................................................................ 171 III-III: Preparation of Adenovirus-MNPs complexes for magnetofection ... 173 III.III.I. Magnetic nanoparticles ............................................................ 175 III.III.II. Virus titration ......................................................................... 180 III.III.III. Adenovirus-MNPs complexes association and stability ....... 184 III.III.IV. Size, Z-potential and magnetophoretic measurements ......... 186 III.III.V. Morphological characterization of the complexes ................. 193 III.III.VI. Complex internalization ........................................................ 198 III.III.VII. Viability assay ..................................................................... 201 III.III.VIII. Infection experiments ......................................................... 203 III.III.IX. Coculture infections .............................................................. 208 III.III.X. In vivo preliminary results ..................................................... 211 III.III.XI. Conclusions........................................................................... 213 IV. Conclusions .................................................................................................. 215 Bibliography .......................................................................................................... 219 Annexe : Theoretical basis of techniques .............................................................. 235 VI.I. Specific Power Absorption ........................................................... 237 VI.II. Dynamic Light Scattering ........................................................... 239 VI.III. SEM ........................................................................................... 241 VI.IV. TEM ........................................................................................... 243 VI.V. Dual Beam .................................................................................. 244 VI.VI. Confocal .................................................................................... 246 4 Abbreviations AC: alternating current AMF: alternating magnetic field APC: antigen presenting cell BSA: bovine serum albumine CD40L: CD40 ligando CPM: counts per minute CSF: cerebrospinal fluid DCs: dendritic cells iDCs: immature dendritic cells mDCs: mature DCs ECs: endothelial cells FBS: fetal bovine serum FCS: fetal calf serum FM: ferro or ferromagnetic FSA: fluorinated surfactant FSC: Forward Scatter G. lucidum: Ganoderma lucidum GFP: green fluorescence protein GM-CSF: granulocyte-macrophage colony-stimulating factor H: magnetic field Hc: coercitivity field IL: interleukin 5 Abbreviations ________________________________________________ INF-γ: interferon γ LPS: Lipopolysaccharides M: magnetization Ms: saturation magnetization Mr: remanence magnetization M-CSF: macrophage colony-stimulating factor MD: multi-domain MFH: magnetic fluid hyperthermia MFI: mean fluorescence intensity MNPs: magnetic nanoparticles MOI: multiplicities of infection MPs: micro particles NPs: nanoparticles MRI: magnetic resonance imaging PBS: Phosphate buffered saline PCS: photon correlation spectroscopy PEI: polyethylenimine PGA: poly-glutamic acid PGE2: prostaglandina E2 PLGA RT: room temperature SD: single-domain
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