Optical Imaging Probe development


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Optical Imaging Probe development

  1. 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. 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. 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. 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. 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. 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. 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. 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
  9. 9. Small organic molecules Current strategies for the development of multifunctional NIR dyes with cancer targeting property 9
  10. 10. Small organic molecules • Direct conjugation a gastric tumor angiogenesis marker candidate Bioconjugate Chem. 2013, 24, 1134−1143 10
  11. 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. 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
  13. 13. Small organic molecules • Target-activatable probe 13
  14. 14. Metal complexes Metal complex? Photophysical Properties M + :nL  M:Ln phosphorescence features luminescence from 10-4 to several seconds. 14
  15. 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
  16. 16. Metal complexes Imaging with lanthanide complexes [Eu2(LC2)3] [Tb2(LC2)3] [Sm2(LC2)3] 16
  17. 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. 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
  19. 19. Metal Complexes • Metal complex-incorporated SiNPs Ru(bpy)3 loaded silica nanoparticles (A) OH–SiNPs (B) COOH–SiNPs (C) PEG–SiNPs Anal. Chem., 2008, 80, 9597 19
  20. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
  34. 34. Quantum Dots (QD) (3) Coordination + hydrophobic dual interaction ligand Advantages Excellent pH stability Highly water-solubility Cheap ligand Relatively compact QD size ~ 16 nm Angew. Chem. Int. Ed. 2008, 47, 3730 34
  35. 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
  36. 36. Metal Nanoclusters Bulky metals 36
  37. 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. 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. 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. 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. 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. 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
  43. 43. Rare earth nanophosphors 43
  44. 44. Rare earth nanophosphors One-Pot Syntheses and Cell Imaging Applications of Poly(amino acid) Coated LaVO4:Eu3+ Luminescent Nanocrystals 44
  45. 45. Design Issues of OI Probes (a) reporter units or payloads Optical properties should be improved for In vivo applications 45
  46. 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. 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. 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. 49. Design Issues of OI Probes (d) Targeting biomolecules Antibody 49
  50. 50. Design Issues of OI Probes (d) Targeting biomolecules Small molecules 50
  51. 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. 52. Design Issues of OI Probes (d) Targeting biomolecules Aptamers 52
  53. 53. Design Issues of OI Probes (d) Targeting biomolecules Aptamers 53
  54. 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
  55. 55. Thank you for kind attention 55