L. D. Carlos

Physics Department & CICECO, Aveiro, Portugal

Luminescent Materials For Biomedical Applications: The

example of Nanothermometers

L. D. Carlos

29/09/2014

Nuno SilvaVitor Amaral

Patricia Lima

Carlos Brites

Duarte Ananias

Mengistie Debasu

João Rocha

Rute Ferreira

Isabel Pastoriza-

Santos

Fernando PalacioAngel Millán

Luiz Marzan

Paulo André

Instituto de Telecomunicações

I. Luminescent materials in bio & nanomedicineI.1 Contrast agents & biomarkersI.2 Nanoparticles for multimodal imaging and theranosticII. Challenges for luminescence in bio & nanomedicineII.1 NIR optical imaging (in vivo and in vitro)II.2 Luminescent nanothermometers III. Why nanothermometry? Which is need for?IV. Ratiometric temperature sensing @ GFHybrids (Aveiro)V. Joining heating and thermometry at the nanoscaleV.1 All-in-one optical heater-thermometer nanoplatform (plasmonic-induced heating)VI. Conclusions

OUTLINE

I. Luminescent materials in nanomedicine

What is luminescence?

“Emission of light by certain materials not resulting from heat.”

…and God said, "Let there be light" (fiat lux), and there …was light!

The Book of Genesis

Why light matters?Central to linking cultural, economic and political aspects of the global society

International Year of Light and Light-based Technologies

UN has recognized the importance of raising global awareness about how light-based technologies promote sustainable development and provide solutions to global challenges in energy, education, agriculture & health.

Light plays a vital role in our daily lives and is an imperative cross-cutting discipline of science in the 21st century

http://www.light2015.org/Home/About.html

Medicine revolution;

XX century telecommunications revolution (laser, laser-diode, optical fiber, Er3+-doped amplifier);

Infrastructure for the Internet

Contrast agents and biomarkers World market reaches more than one billion US dollar

MRI

NMR Imaging (MRI) Contrast Agents Gd chelates, e.g. Gadodiamide, Omniscan Change the relaxation times (T1, T2) of 1H in tissues and body

cavities where they are present

Without CA With CA

Defect of the blood-brain barrier after stroke shown in MRI (T1-weighted images)

Biomarkers

Long (ca. 10-3 s) 5D0 lifetime in the Eu3+ cryptate eliminates the fluorescence interference from other compounds or any unbound XL665.

Concentrations of CD86 and CD28 species are quantify through the intensity of the XL665 luminescence.

Cisbio-US, Inc.

UV Energy transfer

Fluoroimmunoassay Immunological method for clinical diagnosis. Relevant in prenatal and neonatal screening tests, as well as to detect proteins, viruses, antibodies, tumor biomarkers and medicine residues.

Nanoparticles for multimodal imaging and theranostic

M. Ferrari, Nature Rev. Cancer, 2005, 5, 161

The vision: a multifunctional cargo platform

Imaging agents Stimulus sensitive agents Specific targeting moiety Biocompatible polymer Drugs Cell penetrating agents

S. L. C. Pinho et al., Biomaterials, 2012, 33, 925; M. L. Debasu et al., Nanoscale, 2012, 4, 5154

Many examples for bimodal imaging, e.g. MRI & luminescence

Cell internalized SiO2@APS/DTPA:Eu,Gd NPs

T1- & T2-weighted MRI images of cellular pellets

Control (no NPs internalization)

Photos of cellular pellets excited at 393 nm

Cell internalized ϒ-Fe2O3 NPs

positive contrast, r1=4.4 s-1mM-1

negative contrast, T2-shortening Fe2O3 NPs

G. Tian et al., J. Mater. Chem. B, 2014, 2, 1379

Engineered design of theranostic UCNPs Tri-modal imaging & targeted delivery of anticancer drugs

NIR optical imaging

In-vivo multispectral imaging systems(spectral deconvolution filters excitation wavelengths, 390–770 nm, from a white-light source)Mouse also imaged in X-ray mode.

NIR emitting dyes

II. Challenges for PL in nanomedicine

NIR photons penetrate deeper in biological tissues, compared to visible light;

Tissues present less autofluorescence; Better signal-to-noise discrimination; Improved detection sensibility; NIR photons interact less with biological

tissues, reducing the risk of disturbance or damage.

Advantages

http://acs.ufl.edu/?page_id=226

NIR-to-NIR down-shifting PL (1 photon excitation)

G. Chen et al., Acc. Chem. Res., 2013, 46, 1474

Core/shell NaGdF4:Nd3+/NaGdF4 NPs

PL images of HeLa cells incorporated the NPS (λex=740 nm)

In vivo whole body imaging of a mouse subcutaneously injected with the NPs

Depth penetration of light Primary obstacle to applying in-vivo optical molecular imaging (OMI), light

cannot penetrate more than 5-6 cm into human tissue;

In-vivo OMI market will reaches $400 million in 2014

Luminescent thermometers

Temperature measurements are crucial in many scientific investigations and technological developments, 80% of the sensor market throughout the world

Current technological demands (microelectronics, microoptics, photonics, microfluidics, nanomedicine) have reached a point such that the use of conventional thermometry is not able

to make measurements when spatial resolution decreases to the submicron scale (e.g. in intracellular temperature fluctuations and temperature mapping of microcircuits and microfluids).

III. Why nanothermometryWhich is the need for?

J. Lee & N.A. Kotov, Nano Today, 2007, 2, 48; K.M. McCabe & M. Hernandez, Pediatr. Res., 2010, 67, 469; D. Jaque & F. Vetrone, Nanoscale, 2012, 4, 4301; J. Millen et al. Nature Nanotech., 2014, 9 425

Sensing temperature

in an accurate way with sub–

micron resolution

numerous features of micro and nanoscale electronic

devices (thermal

transport, heat

dissipation, and profiles of heat transfer)

critical for understanding

Precise mapping of the temperature of living cells, especially cancer cells strongly improves the perception of their pathology and physiology

Optimization of premature diagnosis and therapeutic processes (e.g. in hyperthermal tumour treatment and photodynamic therapy)

Electron Microscope Photos of Brain Cancer Cells(http://www.alternative-cancer.net/Cell_photo

s.htm)

C.L. Wang et al., Cell. Res., 2011, 21, 1517; G. Kucsko et al., Nature, 2013, 500, 54; N. Inada & S. Uchiyama, Imaging Med., 2013, 5, 303

Increased metabolic activity: Higher T than those of normal tissues

Intracellular temperature distribution

Temperature of living cells is modified during every cellular activity transfer rates as:

cell division

gene expression

enzyme reaction

changes in metabolic activitySTEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY

Lung cancer cell division (SEM)

http://www.sciencephoto.com/set/1336

C.D.S. Brites et al., Nanoscale, 2012, 4, 4799

IV. Ratiometric temperature sensingUnavailability of a nanothermometer with: i) high temperature resolution (< 0.1 degree); ii) ratiometric temperature output; iii) high spatial resolution (< 10–6 m); iv) functional independency of changes in pH, ionic strength and surrounding biomacromolecules; and v) concentration–independent output.

Luminescent thermometers (Ln3+): most suitable class of thermometers to fulfill (simultaneously) those requirements.

How it works?Part of the energy level diagram for Ln3+ aquo

ions Energy separation

between levels comparable to the thermal energy kBT

Impossible to populate a single energy level

Boltzmann statistics: the population will be re-distributed among energy levels with similar energy

|1> is optically populated (from the ground state) Due to the proximity of the |2> level (E), the initial |1>

population is thermally re-distributed among the two levels The |2> population (N2) is (steady-state):

)/(exp12 TkENN B

I1 & I2 are proportional to the corresponding populations:

NCI

I2/I1 ratio:

)/(exp1

2

1

2 TkEC

C

I

IB

ΔE

I2

I1

2

1

depends on geometrical factors and intrinsic properties of the emitting level (e.g. branching ratios and quantum efficiency)

V. Joining heating and thermometry at the nanoscale

ACS Nano, 2014, 8 (5), 5199–5207

Advantages relatively to the dual-particle approach:

Uncontrolled spatial distribution of nanoheaters and nanothermometers

Large distribution of the nanoheater-nanothermometer distances d

Average temperature of the samplevolume under irradiation (emissionintensity includes the contribution of the nanothermometers that are away from the nanoheaters);

Thermal sensing not achieved at the same heating volume.

d

d

Heater-thermometer joint venture at the nanoscale

M. L. Debasu et al., 25, 4868 (2013)

Assess the local temperature of laser-excited Au nanostructures using an all-in-one nanoplatform comprising (Gd,Yb,Er)2O3 nanorods (thermometers) surface-decorated with Au NPs (heaters).

Unambiguous attribution of the white-light emission to an incandescence process.

V.1 All-In-One Optical Heater-Thermometer Nanoplatform

(Gd0.95Yb0.03Er0.02)2O3 NRs: simple wet-chemical route

Synthesis

M. L. Debasu et al., J. Phys. Chem. C, 2011, 115, 15297

Citrate stabilized spherical AuNPs: standard Turkevic methodJ. Turkevich et al., Discuss. Faraday Soc., 1951, 11, 55

Heater-Thermometer Nanoplatforms

I. Pastoriza-Santos et al., Phys. Chem. Chem. Phys., 2004, 6, 5056

NRs-AuNPs-CC (1.25-37.5) nominal Au amount (µmoles of the metal)

AuNPs immobilized on the NRs by the in situ reduction of HAuCl4.3H2O using NaBH4

as a strong reducing agent in aqueous dispersion of the NRs.

The lower the amount of Au precursor, the fewer the number of AuNPs supported on the NRs

Lower Au amount Higher Au amount

TEM IMAGES NRs-AuNPs

Crystallographic planes and interplanar distances for NRs (first image) and AuNPs (second image)

The images on the right side zoom in the regions depicted by the white circles on left.

C = 2.5

C = 1.25

C = 12.5

C = 25

390 585 780 975 1170 1365Norm

aliz

ed A

bso

rban

ce

Wavelength /nm

bare NRs NRs-AuNPs-1.25 NRs-AuNPs-2.5 NRs-AuNPs-5.0 NRs-AuNPs-12.5 NRs-AuNPs-37.5

Localized surface Au plasmon resonance, LSP (aqueous dispersions of NRs-AuNPs-C)

UV-VIS-NIR Absorption

Bare NRs (black lines) and NRs-AuNPs-1.25 (red lines) (600 W.cm-2 excitation with a 980 nm CW laser diode)

Up-conversion emission spectra

∆E≈ 760 cm-1

Yb3+Er3+

2H11/2

4S3/2

4I15/2

2F5/2

2F7/2

980

nm

2F9/2

4I13/2

ET4I11/2

ΔE(2H11/2-4S3/2)≈760 cm-1

limit of no laser excitation (RT)

FIR plot of the 2H11/2→4I15/2 to 4S3/2→4I15/2 transitions vs. laser power density for NRs-AuNPs-C, with C = 0 – 5.0.

Evolution of FIR with pump power

100 200 300 400 500 600

1

2

3

4

bare NRs

NRs-AuNPs-5.0

NRs-AuNPs-2.5

FIR

Laser power density /Wcm-2

NRs-AuNPs-1.25

FIR vs. absolute local temperature

Pump power density32–600 W.cm-2 (1.25)95–455 W.cm-2 (2.5)95–205 W.cm-2 (5.0)

re temperatuysensitivit absolute

(FIR)parameter eric thermometysensitivit relative

TS

S

STS

a

a

Temperature sensitivity

Sensitivity of C=1.25 in the range of physiological interest!

What are the influence of exciting the nanoplataform (through Yb3+) in resonance with the Au surface plasmon?

390 585 780 975 1170 1365Norm

aliz

ed A

bso

rban

ce

Wavelength /nm

bare NRs NRs-AuNPs-1.25 NRs-AuNPs-2.5 NRs-AuNPs-5.0 NRs-AuNPs-12.5 NRs-AuNPs-37.5

How to do this?

Au NRs

Gd2O3:Er/Yb NRs

STEM IMAGES NRs-AuNRs

C = 3.05

in preparation

AuNRs-808nm NRs@PSS@AuNRs-808nm-C=2.28

500 600 700 800 9000.0

0.5

1.0N

orm

aliz

ed A

bsor

banc

e

Wavelength /nm

UV-VIS-NIR Absorption

150 300 450 6000.5

1.0

1.5

2.0

NRs-AuNPs-1.25 NRs@PSS@AuNRs-850nm-1.25

FIR

Laser power density /Wcm-2

360 420 480 540 600

0.5

1.0

1.5

2.0

NRs-AuNPs-1.25 NRs@PSS@AuNRs-850nm-1.25

FIR

Temperature /K

AuNRs have strong heating effect, compared to AuNPs, resonance of the LSP band with the laser beam wavelength.

Distinct dependence of FIR (and temperature) with laser power density (mechanism?)

41

VI. Messages to take home

Luminescent materials play a crucial role in the development of bio and nanomedicine

NIR optical imaging may promote a revolution in the fluorescence microscopy

Heater-thermometer nanoplatforms can improve the efficiency of hyperthermia processes and are exciting tools to study heat transfer processes at the nanoscale (probes to new phenomena?)

THERMOMETRY AT THE NANOSCALEL. D. Carlos & F. Palacio, Eds.

ACKNOWLEDGEMENTS

FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA

PEst-C/CTM/LA0011/2013; PTDC/CTM/101324/2008

EUROPEAN MULTIFUNCTIONAL MATERIALS INSTITUTE

LUMINET— European Network on Luminescent Materials, FP7-PEOPLE-2012-ITN (316906)

COST ACTION MP1202

PVE Grant 313778/2013-2, Science without borders

Spatial averaging > 1.5×103 μm