optical imaging probe development

Probe developments; Optical Imaging Probes

Dr. Chalermchai Pilapong

Center of Excellence for Molecular Imaging (CEMI), Chiang Mai University

SMITH 2013” 28 November 2013, Holiday Inn, Chiang Mai, Thailand.

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Outline

• General Aspects of Optical Imaging (OI) Probe • Organic molecule-based OI Probe

• Metal complex-based OI Probe

• Inorganic nanocrystal-based OI Probe

• Design Issues for probe development

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Optical imaging probes are agents used to visualize, characterize, and

measure biological processes in living systems via emitted light.

Cell tracking and trafficking Live cell imaging In vivo imaging

What is OI Probe?

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Fluorescence is short-lived, with luminescence ceasing

almost immediately (<10-7 sec) ,while phosphorescence

features luminescence from 10-4 to several seconds.

Basic of Luminescence

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Strategy For Wavelength

Selection

Optical imaging window

Visible Near infrared

- minimal light absorption

by tissues and organisms

- Enhanced penetration of both

excitation and emission light

- Improved signal-to-noise ratio

650 – 1000 nm

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Typical sources of OI Probe

Organic molecules (usually with conjugated p-

bonds) – synthetic fluorophores or dye (fluorescein,

rhodamine, …), biological molecules (aromatic

amino acids – Trp, Tyr, chlorophyll, …)

Inorganic nanocrystals – the spectra depend on

the bandgap size, which depends on the size of

the crystal e.g. Quantum dot, metal nanoclusters

Metal complex molecules – transition metal

complex, heavy metal complexes, lanthanide and

actinide ), …

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Small organic molecules

- usually with conjugated π-bonds

- dominate the commercial market of imaging agents,

Abs. 673 nm

Em. 692 nm Abs. 747 nm

Em. 774 nm

Problems - Photobleaching - poor photochemical

stability - very short lifetime

Curr Org Synth. 2011, 8, 521–534

http://www.atdbio.com/img/articles/Cy-dyes-all-large.png


Small organic molecules Newly developed NIR dyes for cancer imaging

- Improved chemical and

photostability

- High fluorescence

intensity

- Long fluorescent life time

- Improved water-soluble

property

Biomaterials 32, 2011, 7127-7138 8


Current strategies for the development of multifunctional

NIR dyes with cancer targeting property

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• Direct conjugation


Bioconjugate Chem. 2013, 24, 1134−1143

a gastric tumor

angiogenesis marker

candidate

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Schematic of different dye labeled nanoparticles

Nano Lett., Vol. 5, No. 1, 2005

Different organic dyes incorporated into silica nanoparticles

• Dye-conjugated nanoparticles


• Dye-conjugated nanoparticles

• Form stable colloidal solutions in a wide variety of in vitro and in vivo environments • Possess chemical stability under a wide variety of physiological conditions (i.e. solvent polarity, reducing environment,ionic strength or pH) • Exhibit limited nonspecific binding to avoid Macrophagocytic system (MPS) uptake • Have programmed clearance mechanisms

• Show good image contrast (high signal-to-noise ratio)

• Have sufficiently long circulation time in the blood if administered intravenously

• Display high sensitivity and selectivity for the target after ligand conjugation

Chem. Soc. Rev., 2012, 41, 2673 12


• Target-activatable probe

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Metal complexes

Metal complex?

M :nL + M:Ln

Photophysical Properties

phosphorescence features luminescence from

10-4 to several seconds.

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Metal complexes

Inorg. Chem. 49 (2010) 2530

Based on phosphorescence not fluorescence

- large Stokes shift (the difference in wavelength

between the absorbed and emitted light)

- long lifetimes

- More resistant to Photodegradable and

photobleaching

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Metal complexes

[Eu2(LC2)3]

[Tb2(LC2)3]

[Sm2(LC2)3]

Imaging with lanthanide complexes

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• Targeted imaging with metal complexes

Metal Complexes

complexes can be modified

routinely through structural

changes of any or all of their

ligands in a stepwise and

potentially combinatorial

approach to synthesis.

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• Targeted imaging with metal complexes

Metal Complexes

Structure of a biotinylated Rh complex

Inorg.Chem. 2010, 49, 4984. Chem. Commun. 46 (2010) 6255

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• Metal complex-incorporated SiNPs

Metal Complexes

(A) OH–SiNPs

(B) COOH–SiNPs

(C) PEG–SiNPs

Ru(bpy)3 loaded silica nanoparticles

Anal. Chem., 2008, 80, 9597 19

• Dye-incorporated SiNPs

Metal Complexes

The methodological comparison between the post-loading route and in situ

co-loading route.

Reverse microemulsion method via hydrolysis/condensation reaction

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• Quantum dots

• Metal nanoclusters

• Rare earth nanophosphors

Luminescence Inorganic

nanocrystals

Why nanoparticles?

1) Drugs, contrast agents,

paramagnetic or

radiolabeled probes can

be vehiculated by NPs

2) NPs can be multi-

functionalized to confer

differents features on them

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Quantum dot; Highly fluorescent semiconductor nanocrystal with a size of ~ 1-10

nm. Its electronic and optical properties deviate substantially from those of the bulk material and are strongly size-dependent

Quantum Dots (QD)

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Quantum Dots (QD)

• Fluorescence emission occurs when an electron excited to the

conduction band returns to the valence band

• The energy of this transition varies with nanoparticle size

• Wavelength of emitted light is also, therefore, size dependent!

5.8 nm 1.2 nm CdSe Quantum Dots 23

Quantum Dots (QD)

Organic fluorophore i.e. fluorescein

-Absorption band narrow:

Limited choice on EX

Long EM tail

Quantum dot -Broad abs :

Wide choice of EX

-EM narrow & symmetric

No EM tail

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• Applications in biological labeling and Imaging

Quantum Dots (QD)

Cell tracking and trafficking

Live cell imaging In vivo imaging

QD has many important applications in biology, especially in cell imaging,

tracking and trafficking as well as in vivo imaging

Nat. Met. 2004, 1, 73

Nat. Comm. 2013, 4, 1619 25

• QDs versus conventional dyes

Quantum Dots (QD)

Nature Biotech., 2003, 21, 41

QD Dye Single QD’s appear 10-20 times brighter than

organic dyes

QD Dye

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Novel Quantum Dot-Based Technique Sees 100 Different Molecules

in a Single Cell

Quantum Dots (QD)

Nat. Comm. 2013, 4, 1619

a multicolour multicycle

in situ imaging technology

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A Novel Clinically Translatable Fluorescent Nanoparticle for Targeted Molecular Imaging of Tumors in Living Subjects

Quantum Dots (QD)

Nano Lett. 2012, 12, 281−286 28

InP/ZnS QD

Synthesis (a) High temperature route e.g. Hot Injection

Technique

Usually requires inert gas (Ar, N2)

Precursor

Generally,

Organometallic cpd

Maintained temperature at ~300 oC for QD growth

Quantum Dots (QD)

S,Se or Te precursor dissolved in

high bp solvent/stabilizing

agent: TOPO, TOP, C11-

amine ~ 340 oC

J. Am. Chem. Soc., 2003, 125, 12567 29

Quantum Dots (QD)

Synthesis (b) Low temperature route Usually requires inert gas (Ar, N2)

QD growth start by heating the solution to 95 oC

Grow for 20, 40 and 90 mins to make green, yellow and red CdTe QDs

Best quantum yield: ~ 45%

Adv. Mater., 2007, 19, 376. 30

Quantum Dots (QD)

Comparison of the two synthetic approaches

High Temperature Route Highly crystalline QD

Mono-disperse, narrow size distribution

Easy core/shell growth control

High quantum yield, up to 90%

QD coated with hydrophobic ligand, insoluble

in water, post surface modification necessary

Low Temperature Route Water-soluble, no post synthesis surface modification

Not highly crystalline

Broader size distribution

Difficult to make core/shell QD

Low quantum yield: typically < 15% (with exceptional ~ 45%)

TEM image

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• Surface modification for QDs

Quantum Dots (QD)

Advantages:

Highly stable, biocompatible,

water soluble QD, high

fluorescence QY maintained

Drawbacks:

Big size, > 20 nm,

Expensive ligand

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Quantum Dots (QD)

Advantages: small QD

size, easy to conduct,

cheap capping ligand

Drawbacks: Quantum yield

decrease, lack of long term

stability, pH-sensitive

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Quantum Dots (QD)

Advantages Excellent pH stability Highly water-solubility

Cheap ligand Relatively compact QD size ~ 16 nm

(3) Coordination + hydrophobic dual interaction ligand

Angew. Chem. Int. Ed. 2008, 47, 3730 34

Quantum Dots (QD)

• QD are a possible replacement for organic dyes

• Quantum Confined systems make scientist can

design the optical properties of the material

• QD have been covalently linked to biorecognition

molecules such as peptides, antibodies, nucleic

acids or small-molecule ligands

• QD have more surface area and functionalities

than conventional dyes; that can be used for

linking to multiple diagnostic and therapeutic

agents

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Metal Nanoclusters

Bulky metals

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Metal Nanoclusters (NCs)

Metals NCs e.g. Au nanoclusters (<2nm)

A simple energy diagram of photoluminescence in gold nanoclusters

the new class of nanomaterials that plays novel physical and chemical properties due to a very small size of this material (< 2 nm)

J. Med Biol Eng., 2009, 29, 276

size-dependent fluorescence

emission, large Stock shift and high

photo-stability. 37

• NCs in bioimaging


Advantages over QD

- low toxicity,

- easy synthesis and

functionalization,

- good water solubility

Sci. Rep, 2013 ,3. 1157; Nanoscale, 2013,5, 1009-1017

Angew. Chem. Int. Ed. 2013, 52, 12572 –12576 38

• Synthesis


1) Using strong reductive agent e.g. NaBH4

-limits their applications in bioimaging and related fields

2) Biomolecular-assisted synthesis - Most common route

- simple and environmental benign

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Renal clearance and Tumor Targeting of Near-IR-Emitting PEG-AuNPs

Scheme of the particle synthesis

In vivo NIR fluorescence images

of the mouse iv injected with PEG-AuNPs

Angew. Chem. Int. Ed. 2013, 52, 12572 –12576

Renal clearance kinetics of the PEG-AuNPs 40


water

37 oC

NIR fluorescent RNase-A-encapsulated gold nanocluster

is used for targeted cellular imaging with potential for oral route administration.

Nanoscale, 2013,5, 1009-1017

Bright-field and the corresponding fluorescence

images of Caco-2 cells after treatment with the

RNase-A-AuNC (a, b) and VB12-R-AuNC (c,

d) for 12 h.

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Inorganic NPs doped with trivalent lanthanide

ions (Ln3+)

Rare earth nanophosphors

Advantages

- narrow emission band widths

(<10 nm)

- large Stokes or anti-Stokes shift

(larger than 100–

200 nm)

- long luminescence lifetimes (ms–s

range),

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One-Pot Syntheses and Cell Imaging

Applications of Poly(amino acid)

Coated LaVO4:Eu3+ Luminescent

Nanocrystals

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(a) reporter units or payloads

Design Issues of OI Probes

Optical properties

should be improved

for In vivo applica-

tions

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(b) Bifunctional chelator or coating reagent

Silica coating Polymer encapsulating

An ideal ligand or chelator should be able to form a stable metal chelate with

high thermodynamic stability and kinetic inertness.


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(c) Linkers; A group of compound used to link between reporter

unit and targeting molecules which can consist of pharmacokinetic

modifers, spacers, conjugation groups

- Minimize non-

specific absorption

- Retain specificity of

targeting molecules


Amine-to-Amine Crosslinkers

Amine-to-Sulfhydryl Crosslinkers

Carboxyl-to-Amine Crosslinkers

Photoreactive Crosslinkers

Sulfhydryl-to-Carbohydrate Crosslinkers

Sulfhydryl-to-Hydroxyl Crosslinkers

Sulfhydryl-to-Sulfhydryl Crosslinkers

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(d) Targeting biomolecules


Identification of lead target candidates Biomarker

discovery • Growth factors (e.g. VEGF, F6F, integrins)

• Membrane receptors stimulated by growth factors

• Intracellular targets (enzymes, steroid receptors)

• Transporters of nutrients and pseudo-nutrients

• Marker associated with change of the extracellular

Matrix (e.g. Metalloproteases)

• Marker associated with the malign formation of the

Cell membrane matrix (e.g. prolin, Cholin)

• Marker of apoptosis

• Marker of vulnerable athorosclerosis plaques (e.g. integrins, LDL)

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Antibody


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Small molecules



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Aptamers are short DNA or RNA oligonucleotides artificially generated to bind

tightly and specifically to various targets including small molecules, protein, cell

and etc..

Advantages of aptamers over antibodies:

• Broader target choice;

• Higher ligand specificity with comparable affinity (nM to pM);

• Produced by chemical synthesis, avoiding using animals and so no batch-

to-batch variations;

• Manufacturing costs and time are all lower compared to that of monoclonal

antibody production.

• More resistant to thermal/chemical denaturation.

• Easy to label with reporters

Nature Rev. Microbiol., 2006, 4, 588 51

Aptamers



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Aptamers



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Summary

Criteria for a useful fluorophore for imaging - able to enter cells;

- localise in desired compartments;

- be biocompatible.e.g. non-toxic and stable/soluble in biological media;

- be excited and emit at non-damaging wavelengths (visible/ NIR);

- show a Stokes shift, or fluorescence lifetime which allows differentiation from

autofluorescence; - be resistant to photobleaching (photochemical destruction of the agent).

Design consideration for Probe development - Sensitivity

- Stability

- Signal-to-noise ratio (SNR)/target-to-nontarget ratio

- Bioavailability

- Biocompatibility

- Pharmacokinetics

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Thank you for kind attention

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optical imaging probe development

Technology

quantum dots qd qd

material qd

probe development2

small qd size

probe metal complex

compact qd size

probe design issues

smallmolecule ligands