Photoluminescent diamond nanoparticles for biolabeling

Domaine du Tremblay nano-MRI Conference 13 July 2010

François TREUSSART, Marie-Pierre ADAM, Orestis FAKLARIS, Abdallah SLABLAB,

Dingwei ZHENG, Loic RONDIN, Géraldine DANTELLE, Jacques BOTSOA, Diep LAI, Vincent JACQUES

and Jean-François ROCH

Laboratoire de Photonique Quantique et Moléculaire,Ecole Normale Supérieure de Cachan & CNRS,

francois.treussart@ens-cachan.fr

Outline

•NV color centers in diamond nanoparticles

•Cellular imaging: nanodiamonds internalization in cell culture

•Drug delivery into cell culture

2

Diamond N-V colour center: photoluminescence

N

VN= NitrogenV= Vacancy

Colour centre creation(electronic/proton irradiation + annealing 800°C)

irradiatednot annealed annealed

HPHT Microcrystal photoluminescence(excitation λ=500-550 nm, exposure time=1.5 s)

not irradiated50 µm

Perfectly photostable at room temperature !

t=106 min t=246 min t=377 mint=0 min

50 µm

50 µm

3A2

3E

1A1metastable

state(s)

488 or532 nm

centrc consists of a

after electron or neutron irradiation.

a vibronic structure in the optical

optical pumping cycle is typically expressed by the

configuration coordinate dcl. The optical transition

of the NV centre occurs tween a singly degenerate

bly degenerate 3E excited

state [ 31. The electric dipole moment of the NV ten

is perpendicular to the symmetric axis of ( 111) [

Fig. 1 shows the absorption, emission and excitation

spectra of the NV centre at 77 K. The zero-phonon line

(ZPL) of the NV centre appears as a sharp line at

637.5 nm with a half width of 4.7 meV which is broad-

ened inhomogeneously due to the internal random strain

[S]. Generally, color centres with a vibronic spectrum

have the possibility to make a laser [6]. The lumines-

cence decay time is one of the important parameters

involved in the lasing of color centres.

The luminescence decay time has been measured in a

* Corresponding author.

09259635/97/$17.00 0 1997 Elsevier Science S.A. All rights reserved. PII SO925-9635(97)00037-X

Fig. 1. The absorption, emission and excitatim spcctt’a of an NV

centre.

number of color centres in dia

are found to be 16.7 ns for the

for the N3 centre [8], 19-23 n

2.55 and 3.1 ns for t e GRl centre [ 10,l I] and

3.2-10.4 ms for the Sl ccntre [ 121. The complex

tion mechanism and internal conversiol~ of energy

the relaxation process after excitation h

cussed [ 8,111. Since type I diamond co

nitrogen atoms as impurity, it is significan

the problem of energy transfer from color centres to the

nitrogen atoms and between color centres, and to study

the complex excitation mechanism. In the NV centre,

H. Hanzawa, et al. Diamond Relat. Mater. 6, 1595 (1997)

T=77 K

3

T=300 K

Photoluminescent Nanodiamonds

4

‣fast irradiation of large quantities of nanoparticles (e-, He+ beam)H.-C. Chang team in Taipei [Y.R. Chang et al. Nature Nanotech. 3, 284 (2008)]

Our team in collab. with CEA-LIST (F. Carel, F.Lainé, Ph. Bergonzo)

‣ irradiation of large quantities of microcrystals and subsequent millingJ.-P. Boudou and P. Curmi (INSERM U829, Evry) [J.-P. Boudou et al. Nanotechnology 20, 235602 (2009)]

Mass production: different approaches

Good marker for bioimaging• size selectable (down to ~5-10 nm)• “easy” surface prefunctionalization⇒ coupling to biomolecule

• perfectly stable photoluminescenceHRTEM image

(A. Thorel & M. Sennour, Centre matériaux, Mines

de Paris, Evry)

5

Production of Photolumin. Nanodiamonds suspensions

Mass production approach developped by Géraldine DANTELLE (PMC Lab. Ecole Polytechnique)

Electron irradiation 13.9 MeV

(collab. F. Lainé, F. Carel, Saphir, CEA Saclay)

Commercial powder (size < 50 nm)(High Pressure High Temp. diamond)

photos JP Boudou

dispersion in water +

strong sonication

Stable aqueous suspension (pH=7)

ς potential -40 mV,carboxylic functions are dominant

Interpretation

air oxydationat 550°C

white powdersp3 surface

vacuumannealing

2 hours, 800°C

black powder

(graphite on surface)

Size + photoluminescence characterization

6

In solution: Dynamic Light ScatteringMean hydrodynamic diam. = 45 nm

10010 size (nm)

12

43

12

4360 nm

AFM PhotoLuminescence

“scanner” piezo. x,y,z

excitationlaser

AFM

After deposition on a substrateCoupled AFM-Scanning confocal setup (Loic Rondin, Abdallah Slablab & Vincent Jacques)

Single Photon counting module

almost all the ND are photoluminescent

100

80

60

40

20

0photo

lum

in N

D (

%)

6040200irradiation duration (min)

Optimizing the brigthness: # NV centers per ND

7

λexc=532 nmPexc=500 µW

“home-built” confocal microscope 80

60

40

20

0Y

(µm

)1086420

X (µm)

4000

3000

2000

1000coups/20 m

s

n=4

1.0

0.8

0.60.4

0.2

0.0corr

elat

ions

nor

mal

isées

-40 -20 0 20retard (ns)

1/n

delay (ns)

norm

alis

ed c

orre

lati

ons

4000

3000

2000

1000

0

coup

s (p

ar 2

0 m

s)

5004003002001000temps (secondes)

perfect photostability

time (seconds)

coun

ts /

20 m

s

filter pinhole50 μm

“scanner” piezo. x,y,z

excitationlaser

dichroic miror

sample

Single-photon detector

time correlation

Single-photon detector

number of colour center/particle ?

HPHT, 35 nm ND

a) b)

c)

15

10

5

0

Y (

µm

)

151050

X (µm)

85

4

4

2

5

5

3000

2500

2000

1500

1000

500

counts

/ 20m

s

8

7

68

9

15

10

5

0

Y (

µm

)

151050

X (µm)

1

2

3

3

23

2

1

4

4

5

3000

2500

2000

1500

1000

500

counts

/ 20 m

s1

52

20

15

10

5

0

occu

rre

nce

3000200010000

counts/ 20 ms

dose 5x1015

H+/cm

2

dose 5x1016

H+/cm

2

Optimizing the brightness of fNDs

8‣O. Faklaris et al., Diam. Relat. Mater. 19, 988 (2010)

HPHT, 35 nm ND

Proton irradiated E=2.4 MeV

(30 µm penetration depth)

Collab. T. Sauvage & M.-F. Barthe, CEMHTI lab.,

Orléans, France

number of color centers/particle

Irrad.Dose(H+/cm2)

mean number of NV center/ND

5.1015 2.7 ± 1.15.1016 7 ± 2.1

[N]~100 ppm, N→NV conversion efficiency ~1.5%

still to be improved...

Bio-imaging applicationNanodiamonds internalisation in

cultured cells

Orestis FAKLARIScollab. Patrick CURMI team, INSERM U829, Univ Evry

Marie-Pierre ADAMcollab. Michel SIMONNEAU team, INSERM U894, Paris

Confocal imaging of ND in HeLa cells

10

Result: nanodiamonds have entered the cells

⇒ are they agregatred or at primary size ?

HeLa cells culture (2 hours incubation

with HPHT 25 nm nanodiamonds)

Phase contrast image of two cells (maybe

after mitosis)

10 µm

nucleus

Photoluminescence raster scans (z-serie)Exc. CW 532 nm (P = 0,5 mW)

Z = 0 (coverslip plane)

Z = +2500 nm

Z = +1500 nm

Z = +3500 nm

Nandodiamond of size~38 nm

Théano Irinopoulou, Institut du Fer à Moulin, INSERM, Paris

Photoluminescence raster scan zoom

11

Raster scan at Z = +1500 nm Time-intensity correlation measurement

⇒ 2 colour centers

Intensity profile

⇒ FWHM 250 nm : diffraction limit

Photoluminescence spectrum

⇒ NVo centres

Zoom

Result: most nanodiamonds are observed at their

primary size inside cell cytoplasm

Results 20 ± 6 % ND-Endosome

ColocalisationFree Nanodiamond in

the cytoplasm, in other compartments ?

Colocalisation of ND and endosomes in HeLa

12

•2 hours incubation with ND (35 nm in size) at 37°C•Early endosome fluo. labelling [Early Endosomal Antigen (EEA)-fluorescein]•Fixed cells

Phase contrast image of HeLa

cells

10 µm

EEA – FluoresceinCollecter 520-560nm

Nanodiamond Photolumin.(laser exc. cw 532 nm)

Merged

Z = +1,5 µm

Z = +2,5 µm

‣ O. Faklaris et al., Detection of single photoluminescent diamond nanoparticles in cell and study of the internalization pathway, Small 4, 2236 (2008)

Blocking endocytosis

13

ND (electron irrad., milled): size~40 nm

very low internalisation

low internalisation

efficient internalisation

2 hours incubation with ND

Further studies ⇒ Receptor mediated

endocytosis

O. Faklaris et al., ACS Nano, 3, 3955 (2009)

at 37°C

phase contrast Photoluminescence(exc. 488 nm, 0.5 mW)

Merged

at 4°C

at 37°C, but cell pretreated with NaN3(ATP production disturbed)

30 µm

30 µm

20 µm

And even more precise ND localisation in cell

14

Collaboration M. SENNOUR and A.THOREL (Ecole des Mines, Centre des matériaux, Evry, France)

HeLa cells, 3 hours incubation with ND

High Res. TEM images

Free nanodiamond

50nm

100nm

nanodiamonds in a vesicle

Zooms

outside cell

inside

ND for neuronal studies

15

Collaboration: Michel Simonneau (INSERM, Paris), Huan-Cheng CHANG (Academia Sinica, Taiwan)

Aim of the project: imaging anomalies in dendritic spines morphology associated to neurodegenerative diseases

lationofafferentfibersprojectingintothefieldofviewwhichisastandardprotocolfortheinductionofLTPinasynapticpathway,whichinducedsimilarchangesasthechemicalLTPprotocol(datanotshown).

Whileitconfirmedpreviousreportsonactivity-dependentsizeincreasesofdendriticspines(5),timelapseimagingusingSTEDrevealeddetailsthatareverydifficulttodetectbyestablishedlightmicroscopyapproaches,suchassubtlechangesintheshapeofspineheads.Infact,duetothelackofresolutionithadnotbeenpossibletoexaminestructuralchangesotherthansimplechangesinspinesize.STEDimagingnowovercomesthislimitationandpermitsstudyingthefinestructureofspinesinlivingtissue.

Fig.4illustratesthetypesofstructuralchangesthatcanbeobservedbySTEDimaging.Theimageswereacquiredbefore(leftmostimage)andaftertheplasticity-inducingstimulationatthetimesindicated.Fig.4showsexamplesofplasticspines,which

changeinsizeandshapeconsiderably.Remarkably,thechangesinshapesusuallyledfromsmallerandamorphousstructurestowardlargerandmoredifferentiatedones,oftentakingoncup-likeshapes.Forexample,Fig.4Cshowsaseriesofimagesofroundspineheadsmorphingintocup-likestructures.Inaddition,Fig.4Dshowsaspinewithalargehead,whichgrowsoutbeforesettlinginanewposition,whereitchangesshapefrombulboustocup-shaped.Whilesomeofthesechangesmayrepresentinstancesofspineheadssplittingintotwoparts,asreportedbyelectronmicroscopy,othersrathersuggestascenariowherebythespinechangesshapeaftertheplasticity-inducingstimulationtocomeintocontactwithitspresumptivepresyn-apticpartner.

TheseexampleshighlightthepotentialofSTEDmicroscopyforlivecellimagingofgeneticallyencodedfluorescencewithintheimportantneurobiologicalcontextofthestructuralplasticityofspinesandsynapses.STEDmicroscopymayevenmakeit

A

000040120200240 0:000:401:202:002:40

C0.8

1alized)

0.2

0.4

0.6

xel intensity (norma

Frame number (20 sec/frame)

0

0.2

05101520

Pix

B

Fig.3.TimelapseSTEDimagingofdendriticstructures.(AandB)SeriesofimageframesofYFP-labeleddendriticspinesacquiredbySTEDmicroscopyat40sec/frame(20frameswereacquiredintotal)underunstimulatedconditions.Arrowsindicatecup-likeshapesofspineheads;STEDpulsepeakintensity400MW/cm2(A)and215MW/cm2(B).(Scalebars,1!m.)(C)Timecourseoffluorescenceintensityintheheadsofspinesasafunctionofconsecutivelyacquiredimageframes(20sec/frame).

Nagerletal.PNAS!December2,2008!vol.105!no.48!18985

NEURO

SCIENCE

dendritic spines

1 µm

adapted from: V. Nägerl et al., «Live cell imaging of dendritic spines by STED microscopy», PNAS 105, 18982 (2008)

primary neuron (from mouse embryo cortex)

5 days of culture on a coverglass(INSERM)

many interconnections established !Phase contrast

ND spontaneous internalization in neurons (1/2)

16

• 35 nm fluorescent ND (from H.-C. Chang, Taiwan) • primary neurons (from mouse embryo cortex) • 20 min incubation time in vitro, and then cultured 5 days on a

coverglass, before fixation

fND PhotoluminescencePhase contrastconfocal rasterscan at z=2.3 µm above the coverglass plane

merged images

5 µm

ND seems not aggregated and localized either in the neuron perinuclear region and in dendrites (to be confirmed by higher resolution imaging)

ND spontaneous internalization in neurons (2/2)

17

5 µm

1

2

3

4

5

6

87

9

z=2.3 µm

increasing height above

the coverglass surface

confocal raster scans

⇒ fND are inside neurons: to be confirmed by histoimmunochemistry labeling of organelles

4000

3000

2000

1000

0

fluo c

ounts

/ 2

0 m

s

3.02.01.00.0

height above the coverglass (!m)

spot1 spot2 spot3 spot4 spot5 spot6 spot7 spot8 spot9

Drug delivery into cultured cells using fNDscollaboration with

A. AlHaddad, J.-R. Bertrand & C. Malvy, Institut Gustave Roussy, VillejuifC. Mansuy & S. Lavielle, Université Pierre et Marie Curie, Paris

G. Dantelle, S. Perruchas & T. Gacoin , Ecole Polytechnique, Palaiseau

H. Girard, C. Gesset & J.-C. Arnault, CEA-LIST, Gif-sur-Yvette

Context: treatment of Ewing sarcoma

19

Children and young adult bone cancer (rare: 200 new cases/year in Europe)

Genetic disease : Chromosomic translocation t(11;22) (q24;q12) ➡ fusion of the EWS gene with the transcription factor gene Fli-1➡ chimeric transcript and chimeric oncogenic protein EWS-Fli-1

expression of EWS-Fli protein responsible for

cell proliferation

Fusion oncogene

Antisense therapeutic strategy using siRNA

20

Principle: Inhibition of EWS-FLI 1 oncogene expression by short interfering RNA (siRNA) targeting and then degrading the corresponding mRNA

mRNApH 5

Endosomal escape

siRNA(negatively charged)

fND(positively charged)

Problem: siRNA does not enter spontaneously into cell delivery ⇒ vehicle necessary

– organic polymer encapsulation– Alternatives: (electrostatic) adsorption onto solid nanoparticle

fluorescence of fND allows to follow the fate of the complex in cell

Adsorption of siRNA on fND

21

Starting materialcarboxylated HPHT fND (size ≈ 35 nm, ζ pot.≈ -40 mV)

fND coated with a polycationic polymer + electrostatic adsorption of the oligonucleotide‣ Polyallylamine (PAH) [S. Vial et al., ChemBioChem 9, 2113 (2008)]

‣ PolyEthyleneImide (PEI800) [X.-Q. Zhang et al., ACS Nano 3, 2609 (2009)]

COO-

Biological efficiency of siRNA:(fND-PEI/PAH) complex

22

Inhibition efficiency• Reference (lipofectamine): 65 %• ND-PEI : 50 %

Inhibition of EWS-Fli1 expression in A673 human cells line

lipofectamine is toxic ⇒ ND is an interesting delivery alternative

0

20

40

60

80

100

120

140

160

180

EW

S-F

Li 1 m

RN

A E

xpre

ssio

n

(% o

f untr

eate

d c

ells

)

cont

rol

ND(P

EI)/

siRNA A

S

ND(P

EI)/

siRNA C

T

ND(P

AH)/s

iRNA A

S

ND(P

AH)/s

iRNA C

T

lipof

ecta

mine/

siRNA A

S

lipof

ecta

mine/

siRNA C

T

1) Measurement of mRNA (Q-PCR)

Contr

ol

ND (P

EI)/

siRNA 1

00

ND (P

EI)/si

RNA 7

5

ND (P

EI)/

siRNA 5

0

ND /s

iRNA C

T

Mol

ecul

ar m

ass

mar

ker

ND

(PA

H)/si

RN

A 5

0

ND

(PA

H)/si

RN

A 2

5

EWS-Fli 1

(68 kDa)

!"#$%&'$()

#"*(

2) Measurement of protein content (Western Blot assay)

What role fluorescence of NV plays ?

23

⇒ strong colocalisation of FITC labelled siRNA and fND proving non

total release...

In progress Imaging siRNA:fND complex by confocal microscopy to monitor siRNA release in cell

10

5

0

-5

-10

Y(!

m)

1050-5-10

X (!m)

525 BP/50 filter

800

600

400

200

co

un

ts / 2

0 m

s

FITC labeled siRNA

10

5

0

-5

-10

Y (!

m)

1050-5-10

X (!m)

645LP filter

500

400

300

200

100

0

co

un

ts / 2

0 m

s

NV fluorescenceNIH 3T3 cell

lines(laser exc. 488 nm)

Summary and prospects✓ ND (size<35 nm) containing NV color centers are bright and

reliable (perfectly photostable) markers

✓ ND are spontaneously internalised in HeLa cell, as individual nanoparticles

‣ Only 20% of internalised ND are trapped in endosomes: the others are free to diffuse in the cytosol

‣ Internalization mechanism is clathrin mediated endocytosis

✓ ND are spontaneously internalized by primary neurons

24

Prospects: superresolution imaging of ND by STED microscopy: application in neurobiology (dendritic spines morphology, dentrite and axon trafficking...)