Magnetic-modified nanodiamond for bio-applications

Chang-You Song 1, Nikolai Perov2, Valentina Bessalova (Samsonova)2, Svetlana Norina2, Li-Chi Liu1, Zhe-Rui Lin1, Yu-Chung Lin1, Ashek-I-Ahmed1, Artashes Karmenyan 1, Olga Levinson3, Boris Zousman3,

Elena Perevedentseva1, Chia-Liang Cheng1,*

1Department of Physics, National Dong Hwa University, Taiwan 2Moscow State University, Russia

3Ray Techniques Ltd., Israel

NDNC 2016, Xi’an China, May 22-26, 2016

1

Nanodiamond use for bioapplications

Carboxylation Functionalization - with linker

2

ND

Carboxylation modifies ND surface with carboxyl groups Carboxylated ND can be functionalized with biomolecules Drug delivery, delivery tracing, targeted interaction, etc.

OO OO

OHAcO

OHO

OAc

H

OCOPh

O

Ph

NH

O

Ph

OH

+

Taxol

Protein- Growth Hormone

Protein- Lysozyme

Protein- X

Drug

ND-X complex

3

RBC

Cancer cell

Bacteria

Zebrafish embryo

Mice

Microorganisms

Nanodiamond use for bioapplications

Combine the properties of ND with magnetism. Applications of NMR, MRI, controlled drug delivery, …

Nanomedicine 2013, 8, 2041-2060

We studied ND interaction with different bio-systems and have found:

Nontoxic / bio-compatible Great property of photoluminescence High efficiency in drug delivery

The Advantages of Magnetic NPs for Bioapplications

L. H. Reddy, et. al., Chem. Rev., 2012, 112 (11), 5818–5878

4

H. Hao et al, J. Mater. Chem. B, 2014, 2, 7978-7987

(1) Drug reservoir (2) Active accumulation

(3) Magnetic Resonance Image

5

• ND powder was treated chemically and thermally in medium free of metals by the method developed by Ray Techniques Ltd. [J. Nanoscience & Nanotechnology, 15(2), 1045-1052 (2015)]

The Magnetic ND (RayND-M)

-10000 -5000 0 5000 10000-1.0

-0.5

0.0

0.5

1.0

M (

em

u/g

)

H (Oe)

DND

RayND-M

• It was found that this specific treatment of ND surface resulted in strong magnetism (ferromagnetism) of diamond nanoparticles. Studies of magnetism in carbon systems – see review paper of T. Makarova 2004, Semiconductors 38, 615–638.

• Energy Dispersive Spectrum RayND-M on Si wafer Magnetic atoms admixtures are negligible: Fe: 0-0.17 weight%, Ni: 0 weight%; Co: 0-0.2 weight%

1000 1500 2000 2500 3000 3500 4000

C-HO-H

Inte

nsit

y (

a.u

.)

wavenumber (cm-1)

Ray ND-M

cND

C=O

Characterization of Magnetic ND (RayND-M): size, surface, structure

0 100 200 300 400 5000

5

10

15

20

Nu

mb

er

(%)

Size (nm)

Average: 90 nm

• Size Distribution: Size: 90 nm.

• SEM image: High aggregation of crystallites is observed. • z-potential: -32.8 mV at pH 7.01.

6

• FTIR spectrum: The –COOH can be observed in RayND-M.

1200 1400 1600 1800

ex: 488 nm

sp2

Inte

nsi

ty (

a.u

.)

Raman shift (cm-1)

sp3

1324 cm-1

1332 cm-1

• Raman spectrum: The shift of 1332 cm-1 to 1324 cm-1 shows that size of crystallites should be small.

Characterization of Magnetic ND (RayND-M): photoluminescence

• The comparison of photoluminescence of Ray ND-M with 5 nm cND.

• It shows the Diamond signal and ZPL with 488 excitation

7

500 600 700 800

641 nm

RayND-M

5 nm cND

ex: 488 nm

Diamond

Inte

ns

ity

(a

.u.)

wavelength (nm)

ex: 532 nm

569 nm

490 500 510 520 530 540 550 560

sp2

Inte

nsit

y (

a.u

.)

wavelength (nm)

sp3

0 1 2 3 4 50

200

400

600

800

1000

Inte

nsit

y (

a.u

.)

Time decay (ns)

2-photon excitation in red and infrared range is done with femtosecond

tunable Ti-Sapphire laser (760 nm, 80 MHz, 140 fs)

0.0 0.5 1.0 1.5 2.0 2.50

1

2

3

4

5

6

7

8

9

10

Nu

mb

er

(10

5)

Time (ns)

Characterization of Magnetic ND (RayND-M): 2-photon excited fluorescence

Fluorescence 2p-lifetime

imaging of RayND-M on

glass substrate

Lifetime decay of RayND-

M luminescence

Histogram of

lifetimes distribution

We observe enough bright 2-photon excited signal, allows 2-photon imaging, in perspective - lifetime and time-gate imaging

Interaction with cells: Cytotoxicity in BHK

(Baby hamster kidney) cell

9

24 hr

contr

ol 1 5 10 50 100

200

0

50

100

150

* * ****

g/mlc

ell v

iab

ilit

y (

%)

24 hr

contr

ol 1 5 10 50 100

200

0

50

100

150

*

g/ml

ce

ll v

iab

ilit

y (

%)

RayND-M

RayND-M + HSA

Some concentration-dependent cytotoxicity is observed The toxicity can be significantly decreased by coating of ND particles surface with albumin (Human Serum Albumin)

Interaction with cells: Fluorescence confocal imaging

Confocal image of BHK cell after interaction with ND for 24 hrs. As Z-scan demonstrates: ND penetrates into cell (and also some ND is attached on the cell membrane). ND is well detectable in cell.

Z-scan:

Excitation Emission

RayND 514 nm 560-600 nm

Nuclei 405 nm 450-490 nm

Cytoplasm 633 nm 640-690 nm

Interaction with cells: 2-photon excitation and fluorescence lifetime imaging

Very good fluorescence signal of RayND-M at 2-photon excitation with femtosecond tunable Ti-Sapphire laser (760 nm, 80 MHz, 140 fs) Makes this ND promising for 2-photon imaging and lifetime imaging :

0 2 4 6 8 10

0

1000

2000

3000

4000

5000

6000

7000

Inte

nsit

y (

a.u

.)

Time delay (ns)

cell autofluorescence

RayND_M

0 2 4 6 8

0

1

2

3

4

5

6

nu

mb

er

(10

4)

Time (ns)

Cell with ND

M-ND

Cell

Lifetime decays allow

distinguish ND and cell signal

BHK cell with RayND-M

Histograms of lifetime distribution

Control without ND autofluorescence of BHK cell

Strong magnetism of nanodiamond is observed and the nanodiamond is characterized for it’s application in bio-medical studies.

The surface and luminescence properties give the possibilities for

biomedical applications.

In the applications magnetic and photoluminescence properties can be combined.

The nature of magnetism is unclear yet and under study.

Summary

12

13

To MOST, Ministry of Science

and Technology of Taiwan

Thank you for your attention!

Acknowledgements:

14

200 300 400 500 600 700 800

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ab

so

rpti

on

Wavelength (nm)

4 5 6 7 8 9 10-40

-35

-30

-25

-20

-15

-10

Zeta

po

ten

tial (m

V)

pH

Tools 1. LakeShore 7407 vibrating sample magnetometer (VSM) (Lake Shore Cryotronics, US), 2. Renishaw, UK, 3. - SNOM, Witec, Germany, 4. Jobin Yvon, T64000, France-Japan 5. Scanning Fluorescence Confocal Microscope (TCS-SP5, Leica, Germany) 6. System for lifetime imaging on the base of femtosecond tunable Ti-Sapphire laser (760 nm, 80 MHz, 140 fs) and PicoQuant scanner Etc…

15

Magnetic properties of carbons and nanocarbons are recently studied.

Pure diamond structure combines diamagnetic and paramagnetic compounds. But in an intermediate graphite–diamond structure spin ordering and magnetic interactions can exist [T. L. Makarova Semiconductors, 2004, 38, 6, 615–638] Ferromagnetic properties of carbon nanostructures are discussed for theoretically predicted structures and are shown experimentally. Particularly, it has been predicted that structures composed of altered C atoms with different hybridization (sp2 - and sp3 –coordinated) can have strong spontaneous magnet moment /under some conditions – strong regular ordering – difficult to realize, but on the micro-level such combination is realized in ND/ Superparamagnetic or/and ferromagnetic particles

Ferromagnetic: M

, arb

. un

it