For in vitro magnetic switching On of apoptosis,Ab–MNPs (1pM)are applied to DLD-1colon cancer cells (1.5×104cells per well),expressing DR4s (Supplementary Section S2),and the cells are placed in between two NdFeB mag-nets (Fig.2c).In the absence of magnetic field,Ab–MNPs are observed as an evenly dispersed weak green fluorescence signal (Fig.2d(i))and remain dispersed over time (Supplementary Section S3).However,large aggregated spots exhibiting a strong green fluorescence signal are observed after the application of a magnetic field for 2h (Fig.2d(ii)red circles and Supplementary Section S4).Scanning electron microscope (SEM)images consistently show that the initial evenly distributed Ab–MNPs change to densely populated aggregates (Fig.2e and Supplementary Section S4).Here,the simulated magnetic field is 0.20T at the centre 28and the average
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MNPs
S
S
N
N
Magnet Magnet
0.0 1 .0
¬1.0¬0.50.5(cm)
0.01 T
t
i ii
5 µm 5 µm
100 nm100 nm Magnet
20 nm Thiolated
MNP
–SH +
O
N
O
O
HN
S
O
O
O
N HN
S
O
O
O
N HN
S
S
N N
Ab¬MNP
Protein A¬MNP
signalling.a,Transmission electron micrograph of15nm zinc-doped
antibody(DR4Ab).A thiolated MNP is linked to protein
The magnetic switch set-up for apoptosis signalling.
magnetic?eld are indicated as a solid line and colour map,
a
M a g n e t i c a l l y i
ii i
ii i ii i
ii
b
body
g
6080
100
a t h (%)
***
Figure 3|In vitro apoptosis induction in the DLD-1colon cancer cell line.a ,Cascade of extrinsic apoptosis signalling pathways.Assembly of DR4s leads to recruitment of DISC,composed of FADD and procaspase-8.Procaspase-8is cleaved to form active caspase-8and leads to subsequent caspase-3
activation.b ,c ,Confocal microscope images of ?uorescently stained active caspase-8(b )and active caspase-3(c )in DLD-1cells for non-activated (i)and magnetically activated (ii)groups.Active caspase-8and caspase-3are immunostained with Alexa-594-labelled secondary antibodies (red)and nuclei are stained with DAPI (blue).d ,e ,Confocal micrographs of membrane inversion (d )and blebbing at the late apoptosis stage (e ),stained with FITC–annexin V (green)and propidium iodide (red),respectively.f ,Differential interference contrast micrographs of DLD-1cell morphology before (i),12h (ii)and 24h (iii)after magnetic ?eld application.Scale bars,10µm.g ,Cell death measured by CCK-8assay.Cells treated with Ab–MNPs (1pM)and magnetic ?eld are compared with the cells alone,Ab–MNPs without magnetic ?eld and TRAIL treatment.Error bars represent standard deviation.(??P <0.01,???P <0.001.)
orders of magnitude weaker and negligible in this set-up where the mid-point of the sample area is 1cm away from the magnet 15(Supplementary Section S5).The Ab–MNPs induce clustering of the DR4s in a similar manner to the biochemical ligand,TRAIL,and they have the unique advantage of being magnetically switched On to activate cell signalling remotely and non-invasively in a spatially and temporally controlled fashion (Fig.2f).
To examine the extrinsic apoptosis signalling process with concurrent assembly of DR4s promoted by using the magnetic switch,biologically important intermediate species of the signalling
cascades are monitored.It is known that clustering of DR4s forms the death-inducing signalling complex (DISC)containing the Fas-associated death domain (FADD)and procaspase-8(refs 20–22).In the DISC,procaspase-8is cleaved to active caspase-8(initiator caspase),which leads to further activation of caspase-3(Fig.3a).Treatment of Ab–MNPs (1pM)on DLD-1cells for 2h with a 0.20T magnetic field results in a strong red fluorescence signal arising from the active caspase-8in the cytoplasm,but no fluorescence is observed in the control group not exposed to a magnetic field (Fig.3b).Subsequent generation of active caspase-3,
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NATURE MATERIALS |VOL 11|DECEMBER 2012|/naturematerials
© 2012 Macmillan Publishers Limited. All rights reserved
Magnet
Magnet Magnet Magnet Magnet
Magnet Magnet Magnet
100 µm
100 µm
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0.01 T
control of apoptosis signalling.Ab–MNPs (1pM)applied to a cell culture slide containing DLD-1cells with DR4s.a ,c ,(c )of magnetic ?eld focused on the cell culture Fluorescence micrographs of the active caspase-3(red)signal nuclei (blue).Each image is created by stitching 50–120?uorescence images pictured by ×200magni?cation confocal microscope.
is strongly amplified in cooperation with other members of the caspase family 31.Entry of the cell into the demolition phase is associated with membrane inversion and blebbing at the late apoptosis stage as major phenomena 17–19.The occurrence of these changes is confirmed by using FITC (fluorescein isothiocyanate)–annexin V and propidium iodide staining,respectively.The observation of a bright fluorescence signal,which is not observed in magnetically non-activated cells,is a clear indicator of this apoptotic outcome (Fig.3d,e).Moreover,temporal morphological changes of the cells are also monitored during the magnetically activated apoptotic process.In comparison with that of normal cells,1.5×104cells treated with Ab–MNPs (1pM;Fig.3f(i))gradually shrink and fragment after 12and 24h application of the magnetic field (Fig.3f(ii,iii)).Quantitative analysis of apoptosis,using a cell counting kit-8(CCK-8)assay,shows that about 52%of the cells treated with Ab–MNPs are dead after magnetic activation,whereas the cells in the non-activated group remain viable (Fig.3g).This level of efficacy is slightly higher than that of TRAIL,the biochemical ligand previously employed as an extrinsic apoptosis signalling agent and potential cancer drug 20–26,which induces cell death in about 45%of the cells treated under the same conditions.We also observe that the cell death rate is dependent on the concentration of Ab–MNPs (Supplementary Section S9).Apoptosis signalling,induced in this manner by the magnetic switch,requires
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