Up-Conversion Nanoparticles for Optical Molecular Imaging Karen Köhler NanoMed Berlin, March 6th,...

Up-Conversion Nanoparticles for Optical Molecular Imaging

Karen Köhler

NanoMed

Berlin, March 6th, 2009

NanoMed 2009Dr. Karen Köhler2009-03-06•

Outline

Application of Photoluminescent Nanoparticles Market Needs

Types of Photoluminescent Nanoparticles

Upconverting Nanophosphors Mechanism

Synthesis

Functionalization

Coupling of Antibodies

Performance Check

Take-Home Message

Intr

od

uct

ion

Mai

n P

art

Su

mm

ary


Polycarbonate

Polycarbonate with QD’s

Polycarbonate

Polycarbonate with QD’sPolycarbonate

Polycarbonate with QD’s

Polycarbonate

Polycarbonate with QD’s Solar cells

Multiplexed biolabeling

Diagnostic imagingOptical fiber-based telecommunications

Light-emitting displays

Solid-state lasersAuthentification

for objects

Photoluminescent Nanoparticles – What for ?

Solar cells


In-vivoIn-vitro

Luminescent Nanoparticles in Diagnostics

Biosensors

POC, Laboratory

Pharmacogenomics personalized medicine

• SNP-analysis

DNA assays• Gene expressions analysis

• Antibody, antigen

• Protein, enzyme

Immuno assays

Improvement of established processes

Higher sensitivity: Less sample material

Smaller: Small size, low-weight instruments

Higher specificity: More reliable

Faster: Shorter response time

More information in a single test: multiplexing

Introduction of new systemsLab-on-a-chip: incl. sample preparation anddetection (DNA, proteins, cells)

Imaging

Migration in cells / organisms (e.g. tumors)

Drug tracking and targeting Contrast- / imaging techniques

Surveillance tasks, e.g.• continuous control of glucose levels, • presence of cancer cells

Control over surface under various conditions (pH, temperature, salt,...)

Specificity

Sensitivity

Clarification of tox issues

introduction of nanoprobes (blood circulation or lymphatic system) and ex-vivo detection


450 500 550 600 650 700 750wavelength, [nm]

ab

so

rpti

on

/em

iss

ion, a

.u.

0.2

0.4

0.6

0.8

1.0

450 500 550 600 650 700 750wavelength, [nm]

450 500 550 600 650 700 750wavelength, [nm]

ab

so

rpti

on

/em

iss

ion, a

.u.

0.2

0.4

0.6

0.8

1.0

ab

so

rpti

on

/em

iss

ion, a

.u.

0.2

0.4

0.6

0.8

1.0

2.5 nm particle size3.8 nm

Physical principle:

crystalline semiconductornanoparticle

h1h2HOMO

LUMO

Crystalline semiconductor nanoparticles

Materials: CdSe, CdS, InAs, InP, …

Size: 1-10 nm just a few 1000 atoms

Confined electron motion leads to altered

optical, physical and chemical properties

Quantum Dots - Baydots®

5.9 nm

Supplier: Bayer Technology Services

ZnS

CdSe

Polymer layer

functional groups

Evident

Design

+


Phosphor Materials

Well established material

TV-screens, fluorescent lamps...)

Doped crystalline particles Size: 1 - 10 µm Host: Y2O3, Y2O2S, LaPO4, ZnS, ...,

Dopants: Lanthanides, transition elements

(Mn, Ag, Cu,...)

Novel synthesis routes for nanophosphor production had to be developed

... defined by dopants not by size


Nanophosphors

CePO4:TbCePO4:TbFirst system:

Individual nanoparticles: ca. 6 - 8 nm (!)

Bright fluorescence (quantum yield up to 50%)

Excitation < 300 nm

Excitation

Emission

0,0E+00

6,0E+06

250 300 350 400 450 500 550 600 650 700

Wavelength / nm

Inte

nsi

ty

Environmentally friendly

20 nm

Low production costs (no necessity for core-shell or size control)

sensitivity ~ 1 fmol

Fulfills the requirements:

Dy3+ Tb3+ Eu3+ Eu2+

Source: Bayer Research Magazine (2004)

Multiplexing capability by variation of lanthanoid ions


Upconversion-Phosphors

NaYF4:Yb,Er

NaYF4:Yb,Tm

Host lattice: fluorides, oxysulfides Dopants: sensitizer (absorber) + fluorescent center (emitter) lanthanides Energy of two or more IR photons is transferred to one lanthanide dopant ion

Excitation: IR Emission: VIS


Upconversion Nanophosphors

Advantages of uc-nanophosphors as biolabels

Minor toxicity Highly stable (photo, thermal, oxidation, shelf-life) Multiplexing capability by variation of lanthanoid ions No photodamage of biological tissues No autofluorescence of tissue

100 nm

IR

NaYF4:Yb,Er NaYF4:Yb,Tm

NaYF4:Yb, Er/Tm Size: 5-100 nm Monodisperse Hexagonal or cubic lattice

Most important applications

Imaging Diagnostics Therapy

S. Heer, K. Kömpe, H. Güdel, M. Haase, Adv. Mat. (2004), 16, 2102.


In-vivo Imaging of uc-Nanophosphors

High laser intensityLow laser intensity


Targeted Nanoparticle-Bioconjugates


Synthesis of Upconversion Nanophosphors

solvent

NH4FNa+

+ solvent HEEDA

Solution IISolution I

uc-nanophosphors dispersed in organic solvent particles have to be water-dispersable for bio applications

HEEDA = N-(2-hydroxyethyl)ethylenediamine

NaYF4:Yb, Er


Surface Functionalization

++

+

++

+

+

-

--

-

-

-- - -

----

-

poly(allylamine hydrochloride)poly(acrylic acid)

poly(ethylenimine)

Polyanion Polycation

+

-- - --

- ---

-

-- +

Electrostatic adsorption of hydrophilic polyelectrolytes on the charged particle surface Choose appropriate polymer and molecular weight for a specific particle type

Ensure colloidal stability under physiological conditions

water dispersableuc-nanophosphors

functional groups for coupling of biomolecules

stable under physiological conditions

-COOH


PEGylation

increase of systemic retention by PEG

Polyethylene glycol

-COOH

-CO

OH

-COOH

-CONH

-CO

NH

-COOH

H2N

EDC, sulfo-NHS

uc-NP@PAA

incomplete reaction with functional PEG leaves carboxylic groups for coupling of biomolecules

Amber tutorials

¯

NanophosphorAntibody

40 nm

PEG-chain


Biofunctionalisation – Test with Streptavidin

Coupling of streptavidin to the carboxylic groups of poly(acrylic acid)

BCA-Assay

Amber tutorials

¯

NanophosphorStreptavidin

(Bicinchoninic acid)

Estimation: ≈ 21 nm2/Streptavidin(maximal loading without PEG)

Protein+

Cu2+

OH-

Cu+ BCA

absorption at 562 nm

Determination of the overall protein amount


0

10000

20000

30000

40000

50000

0 50 100 150 200

concentration of biotin-4- fluorescein / nM

flu

ore

scen

ce in

ten

sity

without SA and nanophosphors

with SA

0

10000

20000

30000

40000

50000

0 50 100 150 200

concentration of biotin-4- fluorescein / nM

flu

ore

scen

ce in

ten

sity

without SA and nanophosphors

with SA

with nanophosphors

Activity of Bound Streptavidin

titration with biotin-4-fluoresceinstreptavidin fluorescence quenching

when bound to SA


The Target

healthy cell tumor cell

target


Development of a Suitable Conjugate

Selection of target structure MUC-1

Selection of a suitable antibody for detection of the target M12

Selection of an optimal fragment for binding / imaging M12-scFv


Coupling of Antibodies Conjugates

EDC/s-NHS-mediated coupling

COOH-functionalized nanophosphor

EDC/s-NHS

M12-scFv conjugatewith His-tag


Proof of Specificity with Quantum Dots - in vitro

QD - M12-scFv - conjugates


Summary

uc-nanophosphors are a new generation of photoluminescent nanoparticles

emission excitation

high application potential in imaging, diagnostics and therapy

synthesis of monodisperse particles

surface functionalization

coupling of antibody conjugates

targeting


Acknowledgement

Stefan Barth

Markus HaaseClaudia Walter

Helga HummelVolker Weiler

Volker Bachmann

Jens WaldeckChristoph Bremer

BMBF Biophotonic II LUNA

Thank you for your Attention !!!Thank you for your Attention !!!

Werner HoheiselBastian BuddeDiana Landen

[email protected]

Up-Conversion Nanoparticles for Optical Molecular Imaging Karen Köhler NanoMed Berlin, March 6th,...

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Transcript of Up-Conversion Nanoparticles for Optical Molecular Imaging Karen Köhler NanoMed Berlin, March 6th,...