Soft magnetic materials: from microsensors to cancer therapy · 2017. 10. 20. · 1 Soft magnetic materials: from microsensors to cancer therapy Alfredo García-Arribas NEW MATERIALS

1

Soft magnetic materials: from microsensors to cancer therapy

Alfredo García-Arribas

NEW MATERIALS FOR SENSORS AND ACTUATORS

DATE: September 28, 2017

TIME: 9:30h – 13:00h

VENUE: Conference Room – Jeronimo de Ayanz Building – C/Tajonar 22 (Public University of Navarra)

ABSTRACT

Scientific and technological breakthroughs in new advanced materials are revolutionizing many industrial and consumer products and are the platforms for continued innovation in many rapidly growing industrial sectors. In particular, many devices are based on new materials properties and processing techniques. These sensors and actuators are being used in environment control, bioelectronics, nanoelectronics, agricultural strategies, automotive industries etc. Besides, these devices should also fit the requirements necessaries to work in several particular conditions. Among others, shape memory alloys, graphene, hybrid and magnetic materials are promising candidates for actual and future applications.

This workshop is held under the financing agreement signed between the Public University of Navarra and the Obra Social La Caixa – CAN Foundation

PROGRAMME

9:30h Opening Iñaki Pérez de Landazabal – Head of INAMAT Ramón Gonzalo – UPNA Research Vice-rector

9:45h Integrative approaches to Inorganic and hybrid Nanomaterials Clement Sanchez. Laboratoire Chimie de la Matière Condensée de Paris UMR ,CNRS, France

10:30 Chemistry of Novel 2D Materials Beyond Graphene Gonzalo Abellán. Univ Erlangen Nurnberg, Department of Chemistry and Pharmacy, Erlangen, Germany

11:15 11:30- Coffee break 11:30 3D printing, a disruptive technology, challenging creativity

Jan Van Humbeeck. Department of Mechanical Engineering (MECH), KU Leuven, Belgium

12:15h Soft magnetic materials: from microsensors to cancer therapy Alfredo Garcia Arribas. Electricity and Electronics Department. Basque Country University (UPV/EHU) and BC Materials, Leioa, Spain .

13:00 Closure Lunch & networking

Registration needed: https://goo.gl/forms/tPQ2rxIDbGOrSYOY2

New materials for sensors and actuators. Pamplona, 28 septiembre 2017

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Outline• Introduction

• Magnetism basics

• Magnetization process

• Soft magnetic materials

• Magnetic microsensors • Magnetic field sensors

• Magneto-impedance sensors

• Electronic compass

• Magnetoelastic sensors • Magnetoelastic effect

• Magnetoelastic resonance

• Oil viscosity sensor

• Magnetic nanodiscs for cancer therapy • Magneto-mechanical actuation

• Magnetic vortex state

• Sub-100 nm vortex discs

• Nanodiscs in cancer cells

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Magnetism Basics

Atomic magnetic moments

Exchange interaction

Spins sum according to certain rules. Only some atoms display net atomic moment.

Magnetism in matter is a quantum effect. Some ingredients are necessary:

Spin of the electronsIt can have only two orientations: up and down.

Only in few materials these atomic moments aligns themselves spontaneously

ferromagnetic antiferromagnetic ferrimagnetic

For applications, only ferromagnetism is useful. Ferrimagnetism is similar.

4

5

Magnetostatic energyA magnetized material produced a strong

external magnetic field.

Huge magentostatic energy.

Magnetism BasicsCrystal anisotropyExchange is isotropic.

Magnetic moments align in selected directions

according to crystal symmetry.

Magnetic domainsThe magnetization is distributed in domains with

different orientations, compatible with anisotropy

to minimize the magnetostatic energy.

The material is macroscopically no-magnetized

6

Magnetism BasicsDomain wallsThe boundary between domains are called domain walls

Domain walls increase the exchange energy.

The equilibrium configuration is an energy compromise.

Magnetic force microscopy

Kerr effect microscopy

7

Magnetization processWhen a magnetic field is applied, the material is magnetized.

Domain wall movement Rotation of the magentization

M is the magnetic moment per unit volume, measured in the direction of the applied field

Hex

M

8

Hysteresis loop

Domain walls are pinned in defects, grain boundaries, etc.

Hex

M

Hc

Mr

HARD magnetic material SOFT magnetic material

9

Magnetic materialsHard magnetic materialsMagnets.

large Mr: produce external magnetic field.

large Hc: are difficult to demagnetise

Soft magnetic materialsLarge permeability.

Low energy loss (slim loop)

µr =dM

dH

Have large response to small magnetic fields.Concentrate and guide the magnetic flux.

Transformers Electric motors

r

10

Soft magnetic materials

Intrinsic propertiesCrystal structure

Magnetic anisotropyTreatmentsCold working

Thermal annealing

….

The softness of a magnetic material is determined by

Grain-oriented silicon steel for transformers

Permalloy Fe20Ni80 has no crystal anisotropy

The softness can be induced only in the direction of interest

-1.0

-0.5

0.0

0.5

1.0

-10 -5 0 5 10

easyhard

Ker

r sig

nal

H (Oe)

HH

easy axis

Permalloy deposited under applied field

11

Soft magnetic materials

crystalline alloy amorphous alloy

Amorphous magnetic alloys (metallic glasses)Topological (and chemical) disorder.

No pinning for domains walls.

No magnetic anisotropy

Extremely soft!

Rapid quenching preparation methodcooling rates of 106 degrees per second Prepared in the form of ribbon or wires

Fe, Co, Ni and B, P, C, Nb, Zr, etc

12















13

Magnetic field sensorsThe great sensitivity to small fields makes soft magnetic materials excellent for magnetic field sensing.

Electronic compassMeasure the Earth magnetic field (about 0.5 G or 50 μT).

Together with accelerometers and gyroscopes provide attitude detection.

Magneto-inductive detection

High resolution.

Poor microelectronic integration.

3-Ax

is D

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des

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oney

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FEA

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esig

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olum

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ssem

bly

Micro flux-gate Anisotropic magnetoresistance (AMR)

Difficult integration.

Noise problems.

Used in some systems

Need of reset field.

Technologies using soft magnetic materials

Both use the change in permeability Use magnetorresistance

14

Magnetic field sensorsHall sensorsWinner technology.

Use Si Hall effect.

Completely integrable (sensor and logic in the same die).

Low sensibility. Need a magnetic concentrator.

The Permalloy concentrator amplifies the magnetic field and guides the perpendicular components into the planar sensors.

15

Magneto-impedance sensors

Magneto-impedance effectSearch for totally integrable solutions with higher sensitivity.

Hiac(!) r

j (A/m2)

δ

δ: penetration depth

Skin effect: the alternating current flows near the surface of the conductor

� =

r2

!�µ

Zmin

ΔZMI (%) = x 100

H

Z

μhigh

μlowZmin

Z

ΔZ

max

16

Z

H

ΔZ

μhigh

μlow

Zmax

Zmin

max. sensibility

Transverse anisotropyH

M

H

Magneto-impedance sensorsMI in planar samples (i.e. thin films)

• Very large sensitivity at low fields in samples with in-plane, transverse anisotropy.

• Need for thick samples to enhance the skin effect

HH

-1.0

-0.5

0.0

0.5

1.0

-10 -5 0 5 10

easyhard

Ker

r sig

nal

H (Oe)

20 nm thick

-1.0

-0.5

0.0

0.5

1.0

-100 -50 0 50 100

easyhard

Ker

r sig

nal

H (Oe)

260 nm thick

well defined in-plane anisotropy development of out-of-plane anisotropy

Problem: softness lost in thicker samples

17


thin (6 nm) Ti spacers

Py layers below critical thickness

~1 μm

-1.0

-0.5

0.0

0.5

1.0

-10 -5 0 5 10

M/M

s

H (Oe)

0

5

10

15

20

-50 -25 0 25 50

MI (

%)

H (Oe)

f = 200 MHz

MI = 23 %

First strategy: increase thickness using non-magnetic spacers

Magnetic softness preserved MI ratio enhancement

A. Svalov et al. APL 100, 162410 (2012)

18

Magneto-impedance sensorsSecond strategy: enhance MI using the magneto inductive effect

Py

Cu

Py/Ti

Py/Ti

MagneticConductive non-magneticMagnetic

Search for best layer configuration:

• thickness of the layers

• thickness ratio of magnetic to non-

magnetic layers

50

100

150

200

250

300

350

1

2

3

4

ΔZ/

Z (%

)

Z ( Ω)

f = 23 MHz

-300

-150

0

150

300

-2

0

2

-30 -20 -10 0 10 20 30

s (%

/Oe)

dZ/dH (Ω

/Oe)

H (Oe)smax = 300%/Oe (2.7 Ω/Oe = 27 kΩ/T)

Extraordinary performance!

[Py(100 nm)/Ti(6 nm)]4 / Cu(400 nm) / [Ti(6 nm)/Py(100 nm)]4

Best performing multilayer structure is

19

• deposited onto Si substrate

• shaped by photolithography

1

20#!m40#!m60#!m80#!m100#!m120#!m140#!m

0.5#mm 1.0#mm 1.5#mm 2.0#mm

width

length

microstrip coplanar

Electronic compass

Micro-sensors


14.5

15.0

15.5

16.0

16.5

17.0

17.5

0 180 360 540 720 900 1080

Z

( Ω)

θ (º)

θSample

Earth field

s = 25 mΩ/º

20















21

Magnetoelastic sensors

MagnetostrictionUses the coupling between magnetic and mechanical properties

Change of length when a magnetic material is magnetized

Used for actuation

• First version of SONAR

• Magnetostrictive

actuators (Terfenol)

Magnetoelastic effectChange in the magnetic state when a magnetic material is deformed

σσ

H = 0

σ > 0σ = 0

produces large permeability changes

~

l

H = 0

l + Δl

H

22

Magnetoelastic sensorsThe magnetoelastic effect can be used to measure deformation, force, torsion, etc.

τ

The detection is non-contact, using pick-up coils to measure permeability changes.

“torductor” ABB

23

Magnetoelastic resonance

h(t) = ho sen ωtAn alternating field excites magneto-elastic waves in the material.

At selected frequencies, mechanical resonances builds up. ε, m, v

ωωr

v(t)

m(t)

!r =n⇡

L

sE

⇢

Young modulus E = E(H): the resonance can be tuned

ωr

HoHoHo

h(t) = ho sen ωt

Ho

24

Magnetoelastic resonanceElectronic article surveillance systems (anti-theft tags)

16 Magnetoelastic Sensors

Acousto-magnetic tag

Plastic sleeve

Magnetostrictive element

Bias magnet

Figure 25. Components of an acousto-magnetic EAS tag. The activeelement is a magnetostrictive amorphous ribbon that is free to oscillateinside a plastic sleeve. The bias magnet is a semi-hard magnetic materialthat can be easily magnetized to activate the tag and demagnetized todeactivate it.

of biasing the sensible element and therefore of selectingits resonance frequency. Figure 26 resumes the operation ofthe system: In the activated state, the bias magnet is mag-netized and the sample resonates at 58 kHz, maintainingan oscillation when the exciting pulse stops. To deactivatethe tag, the authorized operator demagnetizes the bias mag-net (by applying a decreasing-amplitude alternating mag-netic field) so the resonant frequency of the material shiftsto near 60 kHz. The sample only experiments forced oscilla-tions during the existence of the pulse and the receiver doesnot detect any signal between pulses.

Acousto-magnetic EAS systems are working very success-fully around the word and has become a mass application formagnetostrictive amorphous materials. The reliable detec-tion of small tags over large distances in very different con-ditions implies a careful engineering of the sensible material[102, 103].

Figure 26. Principle of operation of an acousto-magnetic EAS system. When the bias magnet is magnetized, it produces a magnetic field Ha on themagnetostrictive element, which resonates at a frequency fa (58 kHz): the tag is activated. In demagnetized state, the field over the element is Hdand its resonance frequency is shifted to fd (about 60 kHz), detuned with the exciting signal: the tag is deactivated.

Other promissing field of sensor development based onthe magnetoelastic resonance is the remote detection ofenvironmental parameters, using the changes of the reso-nant frequency in response to physical paremeters that affectthe magnetostrictive element, such as temperature, stress,pressure, etc. The sensing capabilities of this phenomenaare being actively investigated, and has recently reachedimportant achievements mainly by the work of Grimes andcollaborators. Their investigations and results are compiledin the excellent review “Wireless magnetoelastic resonancesensors: A critical review” [104].

To illustrate the principle of operation of the proposeddevices, let us consider the case of temperature detection.The temperature affects many of the variables that deter-mine the value of the resonance frequency. Apart from thephysical dimensions of the magnetostrictive element, the sat-uration magnetostriction coefficient, the Young’s modulus,the saturation magnetization and the magnetic anisotropyconstants are temperature dependent. All these parametersmake the resonance frequency be dependent on tempera-ture, as is deduced from Eqs. (13) and (14). This depen-dence can be evidenced experimentally: Figure 27 shows theresonance frequency of an amorphous ribbon as a functionof the magnetic field for different temperatures [105]. For agiven value of the applied magnetic field, the resonant fre-quency varies with temperature. According to these results,biasing the resonant element at a given magnetic magneticfield (by means of a suitable polarizing magnet), the sensibil-ity to temperature can be adjusted to different values. Thereis even a compensation point at which the temperature doesnot affect the value of the resonance. The change in theresonant frequency as a function of temperature for differ-ent biasing fields, derived from these results, is presentedin Fig. 28. The use of amorphous ribbons with differentcomposition results in different sensibilities and improved

activated de-activated

Magnetoelastic Sensors 15

Figure 23. Principle of operation of a position sensor based on the magnetostrictive delay line principle. Photographs on the right are differentcommercial versions of the sensor. The housing can be adapted to the specific application, even in a flexible mount. Figures courtesy of MTSSystems Corp. [94].

of exploiting this effect to design useful devices. On theone hand, magnetostrictive materials can be used as tagsor labels for Electronic Article Surveillance (EAS) systems.These tag-and-alarm systems are used, for instance, to avoidunauthorized removal of goods. If the label is activated (inthis context, if it manifests magnetoelastic resonance) whenit crosses an adequate interrogation and detection system, itwill trigger an alarm. Of course, for the system to be useful,a simple method to deactivate the tag, that is, to put it outof resonance, must be provided in case the item be legallytaken out. On the other hand, the magnetoelastic resonancecan be used to monitor any parameter that can affect itsbehavior, usually by producing a shift in the resonancefrequency. Both types of applications, that are reviewednext, share the very convenient feature of allowing remotedetection with suitable means, even through a brick wall orround the corner.

Presently, there are four major technologies used for EASsystems: microwave, magnetic or electromagnetic, radio-frequency and acousto-magnetic [100, 101]. Each one hasdifferent advantages and drawbacks and is more convenientfor a specific situation. The last one is based on the mag-netoelastic resonance and will be described here. In anacousto-magnetic system (Fig. 24), a transmitter emits aradio frequency signal of about 58 kHz in pulses at a rateof 50 to 90 pulses per second. These pulses excite the oscil-lations of the magnetostrictive tag that, if in activated state,resonates exactly at that frequency (58 kHz) and emits a sin-gle frequency signal, like a tuning fork, that is detected by areceiver, triggering an alarm.

In fact, the magnetoelastic oscillations at resonance per-dure when the exciting pulse has finished, so the receiveronly “listen” between pulses. This permits to use signalsof low amplitude (reducing electromagnetic contamination)and to avoid interferences, because the detection is made

when the emitter is off. To deactivate the tag it must beplaced in a situation in which the resonance conditions areno longer met. According to Eq. (14), the resonance fre-quency depends on the Young’s modulus of the material.Fortunately, one of the consequences of the magnetoelas-tic coupling is the !E effect, that is, the dependence ofthe Young’s modulus E on the magnetic state of the mate-rial (Section 2.4). Figure 7 on page 63 shows the depen-dence of the resonance frequency on the external magneticfield. Therefore, if the magnetic state of the magnetostrictiveelement is changed, it no longer resonates at 58 kHz. Theoscillations that are induced by the emitter fade very quicklyafter the exciting pulse has finished and the receiver doesnot detect any signal from the tag. Figure 25 shows the dif-ferent parts of an acousto-magnetic label. The external casecontains a plastic sleeve in which the sensible element, astrip of amorphous metal, is free to oscillate. Also includedis a strip of a semi-hard magnetic material that is responsible

Emitter Receiver

Interrogation zone

Interrogationsignal

Responsesignal

Magnetoelastictag

Figure 24. Electronic Article Surveillance (EAS) system.

25

Magnetoelastic resonanceOil viscosity sensor (for predictive maintenance)

η (cSt)

26















27

Magnetic nanodiscs for cancer therapyMagnetic nanoparticles in bio-medicine

• Magnetic Resonance Imaging (MRI)

• Drug delivery

• Hyperthermia

• …

Iron oxides, chemically produced.

Patterned magnetic particles• Great shape versatility

• Many different compositions

• Excellent reproducibility

• …

Produced by physical methods (vapor deposition, lithography, …)

28

Magneto-mechanical actuation

Kimetal,NatureMaterials9,165–171(2010)

Magneto-mechanical actuation of Permalloy discs with vortex state

29

Magnetic Vortex statePeculiar magnetic behaviour in the nanoscale: no magnetic domains

Vortex state Magnetization process

• Large permeability and magnetization

• Null remanence

high actuation capability

no particle agglomeration

➞

➞

The completion between exchange and magentostatic energy produce different configurations

30

1 µm ∼ 60 nm

intra-cell actuationexternal actuation

Sub-100 nm vortex discsAllow for intra-cellular magneto-mechanical actuation

• Fabrication of sub-100 nm permalloy discs • Magnetic characterization • In-vitro test of magneto-mechanical actuation

31

Sub-100 nm vortex discsFabrication by Hole-mask colloidal lithography

Use charged spheres to create a distribution of holes on a polymer

1) deposit a PMMA layer (spin coating)

2) charge surface with PDDA+ + +

+ + +- - -

3) deposit charged spheres

non-regular dense arrangement of nanospheres

SiPMMA

32

4) Ti sputtering

PMMASi

6) Oxygen plasma PMMA etching

5) Tape stripping

Ti template of holes


33

7) Py sputtering

8) PMMA removal

Py nanodots on Si substrate


34

Sub-100 nm vortex discsDetachment from the substrate

Use of Germanium as sacrificial layer

∅ 60 nm

∅ 140 nm

35

-1.0

-0.5

0.0

0.5

1.0

-120 -80 -40 0 40 80 120

M/MsBB

M/M

s

µ0H(mT)

T=50nmR=70nm

Sub-100 nm vortex discsMagnetic behaviour

59

Additional MFM images were recorded under in-plane magnetic fields to

monitor the displacement of the vortex core towards the edge of the disc. The images

nicely correspond to the magnetic states revealed in the hysteresis loop as shown in

Figure 2.13. At zero applied field, the vortex cores are centred, while the core moves

towards the edge of the disc when a small field is applied, being the vortex core

displacement perpendicular to the field direction. As it can be seen in the loop, the

available fields are not enough to completely expel the vortex and go to a saturated

in‐plane configuration.

Figure 2.13. SQUID hysteresis loop of sample L50, with the imaged points marked by red dots and the corresponding MFM images under different in situ fields.

To complement the experimental results, we performed micromagnetic

simulations in discs of the same geometries. Again, we used OOMMF software and

the material parameters described previously except the cell size. Although, the

usual cell size in this kind of simulations is 4 nm, we obtained the same results with

smaller cell sizes. To increase the density of points in Figure 2.14, the data were

obtained using cell sizes of 1 nm for samples S30 and S50 (R = 30 nm), and of 2 nm

for L30 and L50 (R = 50 nm).

Particularly, we were interested in obtaining the configuration of the

magnetisation in the ground state, at zero applied field, to analyse the structure of

36

Sub-100 nm vortex discsMagneto-mechanical actuation

70

Figure 2.19. Light-transmission experiment to study the mechanical responsiveness of the discs to an external magnetic field.

The experiment consists in applying an intermittent magnetic field (tON and

toff) of 2 mT and 1 Hz and a continuous laser beam passing through the aqueous

solution with discs, while the transmitted light intensity is being recorded. The

magnetic field and the laser beam are in the same direction. The resulting plots,

displayed in Figure 2.20, represent the transmitted light intensity and the applied

magnetic field amplitude as a function of time. The dashed line represents the

current in the Helmholtz coils, i.e., the magnetic field, being the tON period

highlighted with pink bars.

The response of the nanodiscs and the microdiscs is similar: the transmitted

light reaches the maximum intensity when the magnetic field is on, and drops when

switched off. This result can be interpreted as the alignment of the plane of the discs

with the magnetic field, which allows the light to pass through the aqueous solution

and reach the detector, as schematically described in Figure 2.21a. However, the

relaxation time (tR) of the microdiscs is clearly larger than the nanodiscs’. We

37

Nanodiscs in cancer cellsInteraction of microdiscs (R = 1 !m, T = 60 nm)nanodiscs (R = 70 nm, T = 50 nm) with human lung carcinoma cells { 86

Figure 3.1. Protocol of the in vitro assays, from the preparation of the cells (steps 1-4) and the treatment (steps 5 and 6) to the cell viability assessment (step 7). CGM: cell growth medium, SN: supernatant.

All of the assays described hereafter, were performed testing the microdiscs

first because, as they have already been studied, there are some reference conditions

in the literature that are helpful to initiate the experiments.

3.2. Intracellular uptake of discs

To study the internalization of the discs by the cells, images were captured by

fluorescence/brightfield microscopy and, more accurately, by transmission electron

microscopy (TEM). Additionally, we recorded live cell videos over 48 h to monitor

Protocol of the in-vitro assays

38

Nanodiscs in cancer cells

86

Figure 3.1. Protocol of the in vitro assays, from the preparation of the cells (steps 1-4) and the treatment (steps 5 and 6) to the cell viability assessment (step 7). CGM: cell growth medium, SN: supernatant.

All of the assays described hereafter, were performed testing the microdiscs

first because, as they have already been studied, there are some reference conditions

in the literature that are helpful to initiate the experiments.

3.2. Intracellular uptake of discs

To study the internalization of the discs by the cells, images were captured by

fluorescence/brightfield microscopy and, more accurately, by transmission electron

microscopy (TEM). Additionally, we recorded live cell videos over 48 h to monitor

Asses the effect of the discs and the alternating magnetic field

39

Nanodiscs in cancer cells89

Figure 3.4. Micrographs of lung carcinoma cells after 24 h of incubation with (a) microdiscs (R = 1 m and T = 60 nm, covered with gold) and (b) nanodiscs (R = 70 nm and T = 50 nm, covered with gold). The discs were added in nominal proportions of 10 and 2000 particles per cell, respectively.

Regarding the internalization mechanism, the process starts at the

interaction between the particles and the cytoplasmic membrane, possibly through

membrane proteins. Proteins contain a wide range of functional groups, including

alcohols, thiols, carboxylic acids, carboxamides and a variety of basic groups able to

react with the gold surface of the discs. Our hypothesis is that the cells internalize

the particles by endocytosis and are subsequently accumulated into lysosomes,

which are specialized organelles that contain hydrolytic enzymes. Lysosomes

function as the digestive system of cells by processing compounds that enter the cell

from the outside, as well as compounds inside the cell.

Transmission electron microscopy (TEM) was performed to provide insight

to the intracellular localization of the discs. After 24 h of incubation with the

particles, the cells were embedded in an epoxy resin and the solidified sample was

cut in 70-90 nm thick slices (the protocol of the sample preparation is described in

Appendix A). As shown in Figure 3.5, microdiscs are localized inside the cell and

oriented perpendicular to the axis, suggesting that they may have been exposed to a

low magnetic field during the fixation process (the same phenomena was observed

in Ref. [12]). Clearly, the area surrounding the microdiscs contrasts with the texture

of the cytoplasm, indicating they could be encapsulated in a lysosome (the holes are

due to the dragging of the microdiscs during the cutting process).

89

Figure 3.4. Micrographs of lung carcinoma cells after 24 h of incubation with (a) microdiscs (R = 1 m and T = 60 nm, covered with gold) and (b) nanodiscs (R = 70 nm and T = 50 nm, covered with gold). The discs were added in nominal proportions of 10 and 2000 particles per cell, respectively.

Regarding the internalization mechanism, the process starts at the

interaction between the particles and the cytoplasmic membrane, possibly through

membrane proteins. Proteins contain a wide range of functional groups, including

alcohols, thiols, carboxylic acids, carboxamides and a variety of basic groups able to

react with the gold surface of the discs. Our hypothesis is that the cells internalize

the particles by endocytosis and are subsequently accumulated into lysosomes,

which are specialized organelles that contain hydrolytic enzymes. Lysosomes

function as the digestive system of cells by processing compounds that enter the cell

from the outside, as well as compounds inside the cell.

Transmission electron microscopy (TEM) was performed to provide insight

to the intracellular localization of the discs. After 24 h of incubation with the

particles, the cells were embedded in an epoxy resin and the solidified sample was

cut in 70-90 nm thick slices (the protocol of the sample preparation is described in

Appendix A). As shown in Figure 3.5, microdiscs are localized inside the cell and

oriented perpendicular to the axis, suggesting that they may have been exposed to a

low magnetic field during the fixation process (the same phenomena was observed

in Ref. [12]). Clearly, the area surrounding the microdiscs contrasts with the texture

of the cytoplasm, indicating they could be encapsulated in a lysosome (the holes are

due to the dragging of the microdiscs during the cutting process).

Even without functionalization, discs are internalized by the cells

∅ 140 nm

2 µm ∅

∅ 2 !m

40

91

For the further experiments, we used a larger concentration of microdiscs

(nominally 25 microdiscs/cell) to increase the population of cells with particles to

that observed using nanodiscs, i.e., 17 %.

3.3. Cytotoxicity

Prior to the study of the destructive capability of the discs under an external

magnetic field, it is essential to evaluate their cytotoxic effect on the lung carcinoma

cells. For that purpose, we followed the protocol described in section 3.1 and tested

the impact of the microdiscs and the nanodiscs on the vital functions of the cells by

adding, respectively, a nominal proportion of 25 and 2000 discs per cell.

Figure 3.7 collects two representative examples of the cytotoxicity tests. As

described in the point 7 of the protocol, the NucBlue dyes the nuclei of all the cells

blue (Figures 3.7b and 3.7e), which allows us to count the total population when

using the DAPI standard filter. The PI instead, only penetrates in dead cells, thanks

to the lower permeability of the dead cells membrane, and dyes the nuclei red, which

can be observed when using the TRITC standard filter (Figures 3.7c and 3.7f).

Representatively, none of the dead cells, marked with white arrows, have discs as

point the red arrows in the brightfield images (Figures 3.7a and 3.7b). We followed

this procedure in the rest of the assays.

Figure 3.7. Micrographs of lung carcinoma cells after 24 h of incubation with microdiscs (R = 1 m and T = 60 nm, covered with gold) and nanodiscs (R = 70 nm and T = 50 nm, covered with gold). (a, d) Microdiscs and the nanodiscs internalized by the cells. (b, e) The nuclei of all the cells. (c, f) Dead cells (marked with white arrows).

After 24 h incubation, nearly 100% of cells with discs survival

Nanodiscs in cancer cellsCytotoxicity

41

94

Figure 3.10. Live cell video captures at different times. At t = 2 h, the cell marked in red divides. At t = 21 h 30 min, it internalizes a group of microdiscs (orange circle). At t = 34 h 30 min, the cell marked in blue divides, whereas the cell with discs is not able to divide and dies after 42 h of incubation.

3.4. Magneto-mechanical stimulus

The last part of the Thesis addresses the study of the efficacy of the magneto-

mechanically actuated nanodiscs to destroy cancer cells. The magnetic field station

was built based on that reported by D-H Kim et al. [9] and is shown in Figure 3.11a.

The AC magnetic field is generated by pair of Helmholtz coils and the well-plate with

the cells is placed at the centre of the station. The magnetic field amplitude was set

to 10 mT with a frequency of 10 Hz; it was reported to be sufficient to dramatically

damage glioma cancer cell using Permalloy discs with R = 0.5 m in [9].

Figure 3.11. Magnetic field station. Helmholtz coils generate an AC field of 10 mT and 10 Hz (a) parallel or (b) perpendicular to the plane of the coverslips as the red arrows indicate. The well-plate is placed in the centre of the air gap as shown in the inset of the figure b.

94

Figure 3.10. Live cell video captures at different times. At t = 2 h, the cell marked in red divides. At t = 21 h 30 min, it internalizes a group of microdiscs (orange circle). At t = 34 h 30 min, the cell marked in blue divides, whereas the cell with discs is not able to divide and dies after 42 h of incubation.

3.4. Magneto-mechanical stimulus

The last part of the Thesis addresses the study of the efficacy of the magneto-

mechanically actuated nanodiscs to destroy cancer cells. The magnetic field station

was built based on that reported by D-H Kim et al. [9] and is shown in Figure 3.11a.

The AC magnetic field is generated by pair of Helmholtz coils and the well-plate with

the cells is placed at the centre of the station. The magnetic field amplitude was set

to 10 mT with a frequency of 10 Hz; it was reported to be sufficient to dramatically

damage glioma cancer cell using Permalloy discs with R = 0.5 m in [9].

Figure 3.11. Magnetic field station. Helmholtz coils generate an AC field of 10 mT and 10 Hz (a) parallel or (b) perpendicular to the plane of the coverslips as the red arrows indicate. The well-plate is placed in the centre of the air gap as shown in the inset of the figure b.

97

For this reason, the magnetic field station was modified in such a way that the field

direction was perpendicular to the plane of the coverslips, as shown in Figure 3.11b.

Furthermore, the perpendicular configuration increases the magnetic flux density

affecting a cell since, as the A549 cells grow in the XY plane, more magnetic field

lines will cross the cell. Both configurations are schematized in Figure 3.14.

Figure 3.14. a) Cells adhered to the coverslip where most of the microdiscs are horizontally oriented. b) In the parallel configuration (Figure 3.11a), when the field is ON, the discs are already aligned to the field. c) In the perpendicular set-up (Figure 3.11b) the angle between the disc plane and the field direction is larger, enhancing the torque of the particle and the magnetic flux density affecting the cell is also higher (magnetic field lines in red).

Then, in the third assay, the magnetic field was applied perpendicular to the

coverslips plane for 30 min. However, the dead cells rate did not increase from 15 %

(the destruction of some cells is shown in Figure 3.15) indicating that the direction

of the magnetic field is neither the key parameter. The other experimental

conditions we could vary were the amplitude and the frequency of the magnetic

field. As it was demonstrated in Chapter 2, a field as small as 2 mT and 1 Hz was

proved sufficient to cause the mechanical torque of the microdisc in water. However,

the movement of the particles in the cytoplasm is probably restricted and a higher

magnetic field may be required. The field applied in the assays is five times larger

(10 mT and 10 Hz) than that used in the light-transmission experiment, which may

be sufficient to cause the rotation of the discs, but once the they are aligned with the

field direction, the particles could get blocked due to the viscosity of the cytoplasm.

Since our magnetic field set-up cannot produce greater magnetic fields, we did not

97

For this reason, the magnetic field station was modified in such a way that the field

direction was perpendicular to the plane of the coverslips, as shown in Figure 3.11b.

Furthermore, the perpendicular configuration increases the magnetic flux density

affecting a cell since, as the A549 cells grow in the XY plane, more magnetic field

lines will cross the cell. Both configurations are schematized in Figure 3.14.

Figure 3.14. a) Cells adhered to the coverslip where most of the microdiscs are horizontally oriented. b) In the parallel configuration (Figure 3.11a), when the field is ON, the discs are already aligned to the field. c) In the perpendicular set-up (Figure 3.11b) the angle between the disc plane and the field direction is larger, enhancing the torque of the particle and the magnetic flux density affecting the cell is also higher (magnetic field lines in red).

Then, in the third assay, the magnetic field was applied perpendicular to the

coverslips plane for 30 min. However, the dead cells rate did not increase from 15 %

(the destruction of some cells is shown in Figure 3.15) indicating that the direction

of the magnetic field is neither the key parameter. The other experimental

conditions we could vary were the amplitude and the frequency of the magnetic

field. As it was demonstrated in Chapter 2, a field as small as 2 mT and 1 Hz was

proved sufficient to cause the mechanical torque of the microdisc in water. However,

the movement of the particles in the cytoplasm is probably restricted and a higher

magnetic field may be required. The field applied in the assays is five times larger

(10 mT and 10 Hz) than that used in the light-transmission experiment, which may

be sufficient to cause the rotation of the discs, but once the they are aligned with the

field direction, the particles could get blocked due to the viscosity of the cytoplasm.

Since our magnetic field set-up cannot produce greater magnetic fields, we did not

H = 10 mTf = 10 Hz

t = 10, 30 min

Nanodiscs in cancer cellsMagneto-mechanical actuation

42

Nanodiscs in cancer cellsMagneto-mechanical treatment

99

50

60

70

80

90

Cel

l via

bilit

y (%

)

MDs MF || 10 min

MDs MF || 30 min

MDsMF 30 min

NanodiscsMF 30 min

Figure 3.16. Percentages of lung carcinoma cell viability (all of them hold microdiscs or nanodiscs) 4h after the application of the magnetic field of 10 mT and 10 Hz.

Figure 3.17. Micrographs of lung carcinoma cells with nanodiscs (R = 70 nm and T = 50 nm, covered with gold), 4 h after the application of the magnetic field of 10 mT and 10 Hz for 30 min, in the perpendicular configuration. In both examples, the cells that have nanodiscs (marked in red in a and d) have been destroyed (red nuclei in c and f).

To explain the effect of the treatment from the biological point of view, a cell

death mechanism induced by the nanodiscs is proposed and summarized in

Figure 3.18, where the lysosomal membrane rupture is suggested to be the principal

cause. P. Saftig et al. have reported that lysosomal membrane permeabilization can

induce the leakage of lysosomal hydrolases into the cytosol, and lead to cell death

eventually [14, 15]. The membrane of the lysosome is a phospholipidic bilayer as it

Cells with nanodiscs inside die after 30 min in perpendicular field

43

99

50

60

70

80

90C

ell v

iabi

lity

(%)

MDs MF || 10 min

MDs MF || 30 min

MDsMF 30 min

NanodiscsMF 30 min

Figure 3.16. Percentages of lung carcinoma cell viability (all of them hold microdiscs or nanodiscs) 4h after the application of the magnetic field of 10 mT and 10 Hz.

Figure 3.17. Micrographs of lung carcinoma cells with nanodiscs (R = 70 nm and T = 50 nm, covered with gold), 4 h after the application of the magnetic field of 10 mT and 10 Hz for 30 min, in the perpendicular configuration. In both examples, the cells that have nanodiscs (marked in red in a and d) have been destroyed (red nuclei in c and f).

To explain the effect of the treatment from the biological point of view, a cell

death mechanism induced by the nanodiscs is proposed and summarized in

Figure 3.18, where the lysosomal membrane rupture is suggested to be the principal

cause. P. Saftig et al. have reported that lysosomal membrane permeabilization can

induce the leakage of lysosomal hydrolases into the cytosol, and lead to cell death

eventually [14, 15]. The membrane of the lysosome is a phospholipidic bilayer as it

Comparison of the effectiveness of the mechanical treatment

Nanodiscs are more effective!

Nanodiscs in cancer cellsMagneto-mechanical treatment

44

Summary

• Soft magnetic materials enable classical technologies but many new applications are continuously being developed.

• Large permeability is a key parameter for magnetic field sensors.

• Magnetic properties are highly coupled with other effects in soft magnetic materials. For instance,  magneto-elasticity allows several sensing mechanisms.

• Nanotechnology largely benefits from soft magnetic materials. For example, magneto-mechanical actuation of magnetic nanodiscs with vortex state is studied for novel cancer therapies.

45

Grupo de Magnetismo y Materiales Magnéticos

Acknowledgements

Documents

Soft magnetic materials: from microsensors to cancer therapy · 2017. 10. 20. · 1 Soft magnetic materials: from microsensors to cancer therapy Alfredo García-Arribas NEW MATERIALS