Objetivo 6 Análisis de la influencia de de los mecanismos de internalización de NPM

en diferentes líneas celulares tumorales sobre sus propiedades magnetotérmicas

Actividad 2 Estudios de internalización y distribución intracelular de NPMs

Grupo GBECNB José L. Carrascosa

Superparamagnetic OD15 Vs MCF-7

MCF7 + [0,5μg/μl] SPIONs

24h

MCF7 human breast cancer cells

control

Calero, M. Chiappi, M. et al., J.Bionanotech. , 2015

≤200 nm CLATHRIN-MEDIATED

ENDOCYTOSIS

Macropinosome

Early endosome

Late endosome (multivesicular bodies)

Lysosome

Clathrin-coated vesicles

200 nm

200 nm

MACROPINOCYTOSIS

200 nm

200 nm

GOLGI NETWORK

pH 6.2

pH 5.2

Calero, M. Chiappi, M. et al., J.Bionanotech. , 2015

Cryo-Soft X-ray tomography

SXT

Supe

r-Re

solu

tion

Conf

ocal

cryo-ET

SXT

Resolution in Z (nm)

Thic

knes

s (μm

)

ALBA - MISTRAL

Xray-source

CCD

Zone Plate Objective diffractive lens

Central stop

Capillary condenser

BESSY II - U41-TXM

Cryo-Soft X-ray tomography

CRYO EPI-FLUORESCENT microscopy cryo-stage

XRAY CRYO-TOMOGRAPHY

MCF7 CELL CULTURE+SPIONs

ON FINDER GRIDS & Plunge freezing

BESSY II U41-TXM Grid design

ALBA MISTRAL EM Grid design

Soft X-ray cryo-tomography Workflow

Plunge freezing

10x transmited light on-line

VLM-SXTM Correlative workflow

• There is an Increase in volume occupied by endocytic organelles containing SPION • The nanoparticles travel from the exterior to a perinuclear area • The size of the endocytic organelles containing SPION increases with incubation time, but cellular division

alters this progression

Cryo-Soft X-ray tomography

Energy Stack: 700 to 730 eV

700 eV; Pre-edge 709 eV; L3 iron edge 720 eV; L2 iron edge 730 eV; Post-edge

O.D.

700 eV 709 eV

Ratio (709/700) projection image

Iron chemical map (3D volume)

2D projection images

X-ray mosaic in vivo fluorescent

2 µm 2 µm

8 µm 25 µm

Near Edge Absorption Soft X-ray Tomography

Conesa JJ. et al., Scientific Reports (2016)

2 µm 500 nm

Conesa JJ. et al., Scientific Reports (2016)

Near Edge Absorption Soft X-ray Tomography

Structure of Macromolecular Complexes José L. Carrascosa Javier Chichón María José Rodríguez Michele Chiappi Javier Conesa Unidad de Biocomputación Carlos O. Sanchez Joaquín Oton

ALBA, Barcelona Eva Pereiro Instituto de Ciencia de Materiales de Madrid Puerto Morales IMDEA Nanociencia Gorka Salas