Secure Zero Touch enabled Edge compute with Dell NativeEdge via FDO _ Brad at...
Optical Imaging Probe development
1. Probe developments;
Optical Imaging Probes
Dr. Chalermchai Pilapong
Center of Excellence for Molecular Imaging (CEMI), Chiang Mai University
1
SMITH 2013” 28 November 2013, Holiday Inn,
Chiang Mai, Thailand.
2. 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
2
3. What is OI Probe?
Optical imaging probes are agents used to visualize, characterize, and
measure biological processes in living systems via emitted light.
Live cell imaging
Cell tracking and
trafficking
In vivo imaging
3
4. Basic of Luminescence
Fluorescence is short-lived, with luminescence ceasing
almost immediately (<10-7 sec) ,while phosphorescence
features luminescence from 10-4 to several seconds.
4
5. Strategy For Wavelength
Selection
Optical imaging window
Visible
Near infrared
650 – 1000 nm
- minimal light absorption
by tissues and organisms
- Enhanced penetration of both
excitation and emission light
- Improved signal-to-noise ratio
5
6. Typical sources of OI Probe
Organic molecules (usually with conjugated pbonds) – synthetic fluorophores or dye (fluorescein,
rhodamine, …), biological molecules (aromatic
amino acids – Trp, Tyr, chlorophyll, …)
Metal complex molecules – transition metal
complex, heavy metal complexes, lanthanide and
actinide ), …
Inorganic nanocrystals – the spectra depend on
the bandgap size, which depends on the size of
the crystal e.g. Quantum dot, metal nanoclusters 6
7. Small organic molecules
- usually with conjugated π-bonds
- dominate the commercial market of imaging agents,
Abs. 673 nm
Em. 692 nm
Curr Org Synth. 2011, 8, 521–534
Abs. 747 nm
Em. 774 nm
Problems
Photobleaching
poor photochemical
stability
very short lifetime
7
8. 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
10. Small organic molecules
• Direct conjugation
a gastric tumor
angiogenesis marker
candidate
Bioconjugate Chem. 2013, 24, 1134−1143
10
11. Small organic molecules
• Dye-conjugated nanoparticles
Schematic of different dye labeled nanoparticles
Different organic dyes incorporated into silica nanoparticles11
Nano Lett., Vol. 5, No. 1, 2005
12. Small organic molecules
• Dye-conjugated nanoparticles
• Form stable colloidal solutions in a wide
• Show good image contrast (high signal-to-noise
variety of in vitro and in vivo environments
ratio)
• Possess chemical stability under a wide variety • Have sufficiently long circulation time in the
of physiological conditions (i.e. solvent polarity,
blood if administered intravenously
reducing environment,ionic strength or pH)
• Display high sensitivity and selectivity for the
• Exhibit limited nonspecific binding to avoid
target after ligand conjugation
Macrophagocytic system (MPS) uptake
• Have programmed clearance mechanisms
12
Chem. Soc. Rev., 2012, 41, 2673
15. Metal complexes
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
Inorg. Chem. 49 (2010) 2530
15
17. Metal Complexes
• Targeted imaging with 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.
17
18. Metal Complexes
• Targeted imaging with metal complexes
Structure of a biotinylated Rh complex
Inorg.Chem. 2010, 49, 4984.
Chem. Commun. 46 (2010) 6255
18
20. Metal Complexes
• Dye-incorporated SiNPs
Reverse microemulsion method via hydrolysis/condensation reaction
The methodological comparison between the post-loading route and in situ
co-loading route.
20
21. Luminescence Inorganic
nanocrystals
• Quantum dots
• Metal nanoclusters
• Rare earth nanophosphors
Why nanoparticles?
1) Drugs, contrast agents,
paramagnetic or
radiolabeled probes can
be vehiculated by NPs
2) NPs can be multifunctionalized to confer
differents features on them
21
22. Quantum Dots (QD)
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
22
23. 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
24. 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
24
25. Quantum Dots (QD)
• Applications in biological labeling and Imaging
Live cell imaging
Cell tracking and
trafficking
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
26. Quantum Dots (QD)
• QDs versus conventional dyes
QD
Dye
Single QD’s appear 10-20 times brighter than
organic dyes
Dye QD
Nature Biotech., 2003, 21, 41
26
27. Quantum Dots (QD)
Novel Quantum Dot-Based Technique Sees 100 Different Molecules
in a Single Cell
a multicolour multicycle
in situ imaging technology
Nat. Comm. 2013, 4, 1619
27
28. Quantum Dots (QD)
A Novel Clinically Translatable Fluorescent Nanoparticle for Targeted
Molecular Imaging of Tumors in Living Subjects
InP/ZnS QD
Nano Lett. 2012, 12, 281−286
28
29. Quantum Dots (QD)
Synthesis (a) High temperature route e.g. Hot Injection
Technique
Usually requires inert gas (Ar, N2)
Precursor
Generally,
Organometallic cpd
S,Se or Te precursor dissolved in
high bp solvent/stabilizing
agent: TOPO, TOP, C11amine ~ 340 oC
Maintained temperature at ~300 oC for QD growth
J. Am. Chem. Soc., 2003, 125, 12567
29
30. 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
31. 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
TEM image
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%)
31
32. Quantum Dots (QD)
• Surface modification for QDs
Advantages:
Highly stable, biocompatible,
water soluble QD, high
fluorescence QY maintained
Drawbacks:
Big size, > 20 nm,
Expensive ligand
32
33. Quantum Dots (QD)
Advantages: small QD
size, easy to conduct,
cheap capping ligand
Drawbacks: Quantum yield
decrease, lack of long term
stability, pH-sensitive
33
35. 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
35
37. Metal Nanoclusters (NCs)
Metals NCs e.g. Au nanoclusters (<2nm)
the new class of nanomaterials that plays
novel physical and chemical properties due
to a very small size of this material (< 2 nm)
A simple energy diagram of photoluminescence
in gold nanoclusters
J. Med Biol Eng., 2009, 29, 276
size-dependent fluorescence
emission, large Stock shift and high
photo-stability.
37
38. Metal Nanoclusters (NCs)
• 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
39. Metal Nanoclusters (NCs)
• 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
39
40. Metal Nanoclusters (NCs)
Renal clearance and Tumor Targeting of Near-IR-Emitting PEG-AuNPs
Scheme of the particle synthesis
Renal clearance kinetics of the PEG-AuNPs
Angew. Chem. Int. Ed. 2013, 52, 12572 –12576
In vivo NIR fluorescence images
of the mouse iv injected with PEG-AuNPs
40
41. Metal Nanoclusters (NCs)
NIR fluorescent RNase-A-encapsulated gold nanocluster
is used for targeted cellular imaging with potential for oral route administration.
37 oC
water
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.
Nanoscale, 2013,5, 1009-1017
41
42. Rare earth nanophosphors
Inorganic NPs doped with trivalent lanthanide
ions (Ln3+)
Advantages
- narrow emission band widths
(<10 nm)
- large Stokes or anti-Stokes shift
(larger than 100–
200 nm)
- long luminescence lifetimes (ms–s
range),
42
44. Rare earth nanophosphors
One-Pot Syntheses and Cell Imaging
Applications of Poly(amino acid)
Coated LaVO4:Eu3+ Luminescent
Nanocrystals
44
45. Design Issues of OI Probes
(a) reporter units or payloads
Optical properties
should be improved
for In vivo applications
45
46. Design Issues of OI Probes
(b) Bifunctional chelator or coating reagent
An ideal ligand or chelator should be able to form a stable metal chelate with
high thermodynamic stability and kinetic inertness.
Silica coating
Polymer encapsulating
46
47. Design Issues of OI Probes
(c) Linkers; A group of compound used to link between reporter
unit and targeting molecules which can consist of pharmacokinetic
modifers, spacers, conjugation groups
-
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
Minimize nonspecific absorption
Retain specificity of
targeting molecules
47
48. Design Issues of OI Probes
(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)
48
49. Design Issues of OI Probes
(d) Targeting biomolecules
Antibody
49
50. Design Issues of OI Probes
(d) Targeting biomolecules
Small molecules
50
51. Design Issues of OI Probes
(d) Targeting biomolecules
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 batchto-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
52. Design Issues of OI Probes
(d) Targeting biomolecules
Aptamers
52
53. Design Issues of OI Probes
(d) Targeting biomolecules
Aptamers
53
54. 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
54