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7 imaging


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7 imaging

  1. 1. Imaging in vitro
  2. 2. Quantum dots as tools for cellular imaging
  3. 3. What are quantum dots ? • Crystalline fluorophores • CdSe semiconductor core • ZnS Shell
  4. 4. What are quantum dots• Unique Spectral properties – Broad absorption – Narrow emission – Wavelength depends on size• Hydrophobic crystals 3 nm
  5. 5. Making hydrophobic quantum dots bio-compatible• Various methods for making them water-soluble – Derivatizing surface with mercaptoacetic acid – Encapsulating in phospholipid micelles or liposomes – Coating them with amine-modified polyacrylic acid
  6. 6. Conjugating quantum dots to biomolecules Avidin• Avidin or protein-G with positively charged tail conjugated to negatively charged DHLA coat of quantum dots protein G
  7. 7. Quantum dots v/s other fluorescent probesPhotostability (quantum dots do not photobleach) Wu et al. 2003 Red: qdot 605 Conjugate Green: Alexa488 Conjugate
  8. 8. Quantum dots v/s other fluorescent probes• Broader excitation spectrum and narrower gaussian emission spectrum• No spectral overlap between dots of different size – better for multiplexing Jaiswal & Simon 2004
  9. 9. Quantum dots v/s other fluorescent probes• Brighter than other fluorophores Quantum dots Fluorescein Larson et al. 2003
  10. 10. Quantum dots and imaging Quantum dots FITC-Dextran In vivo visualization of capillaries Larson et al. 2002
  11. 11. Quantum dots and imagingCancer cell surface marker red & green Microfilaments Actin filaments Nuclear antigens Wu et al. 2003
  12. 12. Quantum dots and imaging Diffusion of single Qdot-GlyRs in synaptic boutonsGlycine Receptors Dahan et al. 2003
  13. 13. Quantum dots and imaging EGF-QDEGF receptor Live imaging of receptor mediated endocytosis Lidke et al. 2004
  14. 14. Quantum dots and imaging Individual vesicles - Dendritic cell processesTemperature variations Observing high resolution structure in dendritic cells
  15. 15. Quantum dots and imaging 1 m 200 nm 200 nmSingle quantum dot crystals can be observed in electron micrographs
  16. 16. Quantum dots and imaging• Quantum dots have been used in FRET• In conjunction with Texas Red• In conjunction with fluorescent quenchers Willard et al. 2003
  17. 17. QDOTS IN VIVO
  18. 18. Advantages• Specific labeling of cells and tissues• Useful for long-term imaging• Useful for multi-color multiplexing• Suitable for dynamic imaging of subcellular structures• May be used for FRET-based analysis
  19. 19. Disadvantages• Colloidal polymer-coated quantum dots can aggregate irreversibly• Toxic in vivo• Quantum dots are bulkier than many organic fluorophores – Accessibility issues – Mobility issues• Cannot diffuse through cell membrane – Use of invasive techniques may change physiology
  20. 20. Imaging in vivo
  21. 21. X-raydense,fluorescent,metallic,or magnetic cores
  22. 22. Multifunctional NanoparticlesFor imaging 25
  23. 23. X-ray CTCT is ubiquitous in the clinical setting as. The increasing use anddevelopment of micro-CT and hybrid systems that withPET, MRI.The most investigated NPs in this field are gold NPs, since theyhave large absorption coefficients against the x-ray source usedfor CT imaging and may increase the signal-to-noise ratio of thetechnique.To date, different types of gold NPs have been tested in apreclinical setting as contrast agents for molecular imaging:nanospheres, nanocages, nanorods and nanoshells. Gold NPsformulations as an injectable imaging agent have been utilized tostudy the distribution in rodent brain ex vivo
  24. 24. nanocage nanorod nanosphere nanoshell Size 4-40 nm
  25. 25. MRIseveral nanotechnological approaches have been devised, based on the idea ofcarrying a substantial payload of Gd chelates. Examples include liposomesmicelles dendrimers fullerenes. However, this approach has not yet achievedclinical applications.To this end, magnetic NPs (MNPs) are of considerable interest because theymay behave either as contrast agents or carriers for drug delivery. Among these,the most promising and developed NP system is representedbysuperparamagnetic iron oxide agents
  26. 26. PETThe strategy utilized is consisting in incorporatingPET emitters within the components of the NP, orentrapping them within the core. Oku et al (2011)employed PET to image brain cancer using positron-emitting labelled liposomes in rats. Plotkin et al(2006) used PET radioisotopes for targeting the intra-tumourally injected magnetic NPs in patients withglioblastoma.
  27. 27. Magnetic Nanoparticles 31
  28. 28. Types of Magnets• Ferromagnetic materials: the magnetic moments of neighboring atoms align resulting in a net magnetic moment.• Paramagnetic materials are randomly oriented due to Brownian motion, except in the presence of external magnetic field. 32
  29. 29. Superparamagnetic• Combination of paramagnetic and ferromagnetic properties – Made of nano-sized ferrous magnetic particles, but affected by Brownian Motion.• They will align in the presence of an external magnetic field.
  30. 30. The most promising and developed NP system isrepresented by superparamagnetic iron oxideagents, consisting of a magnetite (Fe3O4) and/ormaghemite (Fe2O3) crystalline core surrounded by alow molecular weight carbohydrate (usually dextranor carboxydextran) or polymer coat.. Iron oxide NPs can be classified according to theircore structure, such as Monocrystalline (MION;10–30 nm diameter), or according to their size asultra-small superparamagnetic (USPIO) (20–50 nmdiameter), superparamagnetic (SPIO) (60–250 nm).
  31. 31. Dextran Coated Magnetite Nanoparticles• Synthesis of polysaccharide covered superparamagnetic oxide colloids – For MRI imaging• FDA max size for injectables = 220 nm.• Smaller sizes (<100 nm) have longer plasma half-life. – Blood clearance by Reticuloendothelial system (RES) – Liver and Spleen• Without coating, opsonin proteins deposit on Magnetite and mark for removal by RES
  32. 32. Formation of Nanoparticles• Solution of Dextran and Ferric hexahydrate (acidic solution) – Less Dextran Larger Particles• Drip in Ammonium hydroxide (basic) at ~2oC• Stirred at 75oC for 75 min.• Purified by washing and ultra-centrifugation• Resulting Size ~ 10-20 nm• Plasma half-life: 200 min 37
  33. 33. Variation of Formation• Change Coating Material – Various other starches, Sulfated Dextran (for functionalization)• Crosslinking coating material – Increases plasma half-life – Same Particle Size 38
  34. 34. Magnetite Cationic Liposomes (MCL) Fe3O4• Why Cationic? – Interaction between + liposome and – cell – membrane results in 10x uptake.
  35. 35. Formation of MCL• magnetite NP dispersed in distilled water• N-( -trimethyl-amminoacetyl)-didodecyl-D- glutamate chloride (TMAG) Dilauroylphosphatidylcholine (DLPC) Dioleoylphosphatidyl-ethanolamine (DOPE) added to dispersion at ratio of 1:2:2• Stirred and sonicated for 15 min• pH raised to 7.4 by NaCl and Na phosphate buffered and then sonicated
  36. 36. Uses of Nano Magnets• Hyperthermia – An oscillating magnetic field on nanomagnets result in local heating by (1) hysteresis, (2) frictional losses (3) Neel or Brown relaxation
  37. 37. Cancer Treatment • Heating due to magnetic field results in two possibilities Death due to overheating Increase in heat shock proteins result in anti-cancer immunity. 44Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328
  38. 38. Delivery Magnetic nanoparticles • Magnetite nanoparticles encapsulated in liposomes – (1) Antibody conjugated (AML) – (2) Positive Surface Charge (MCL) • Sprague-Dawley rats injected with two human tumors. – Liposomes injected into 1 tumor (black) and applied Alternating Magnetic FieldIto A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328 45
  39. 39. Effect of HyperthermiaTreatedTumor Before TreatmentUntreatedTumor Rectum• Non-local heating in body is the result of eddy-currents – The currents resulting from the After Treatment magnetic field produce heat
  40. 40. Uses of Nano Magnets• MRI imaging.Iron oxide agents shorten T2 and T2* relaxation times on T2-and T2*-weighted MRI images, creating low signal ornegative contrast. They can also be detected by MRI withT1, off resonance, and steady-state free precession sequences
  41. 41. Uses of Nano Magnets• External Magnetic field for nanoparticle delivery – Magnetic nanoparticles loaded with drug can be directed to diseased site for Drug Delivery or MRI imaging.
  42. 42. Magnetic Drug Delivery System• Using Magnetic Nanoparticles for Drug Delivery • Widder & others developed method in late 1970s • Drug loaded magnetic nanoparticles introduced through IV or IA injection and directed with External Magnets • Requires smaller dosage because of targeting, resulting in fewer side effects 51
  43. 43. Magnetic Nanoparticles/Carriers M M• Magnetite Core M• Starch Polymer Coating M Magnetite Core M • Bioavailable Starch Polymer • Phosphate in coating for functionalization M• Chemo Drug attached to Coating M • Mitoxantrone• Drug Delivered to Rabbit with Carcinoma 52
  44. 44. Results of Drug Delivery• External magnetic field (dark) • deliver more nanoparticles to tumor• No magnetic field (white) • most nanoparticles in non tumor regions 53
  45. 45. Magnetic nanoparticles in medicineThey consist of a metal or metallic oxide core, encapsulated inan inorganic or a polymeric coating, that renders the particlesbiocompatible, stable, and may serve as a support forbiomolecules.• Drug or therapeutic radionuclide is bound to a magneticNP, introduced in the body, and then concentrated in the targetarea by means of a magnetic field.• Depending on the application, the particles release the drug orgive rise to a local effect (hyperthermia).• Drug release can proceed by simple diffusion or take placethrough mechanisms requiring enzymatic activity or changes inphysiological conditions (pH, osmolality, temperature, etc…).
  46. 46. Multifunctional Magnetic Nanoparticles• Magnetic nanocrystals as ultrasensitive MR contrast agents: MnFe2O4• Anticancer drugs as chemotherapeutic agents: doxorubicin, DOX• Amphiphilic block copolymers as stabilizers: PLGA-PEG• Antibodies to target cancer cells: anti-HER antibody (HER, herceptin) conjugated by carboxyl group on the surface of the MMPNs 55 Yang, etal. Angew. Chem. 2007, 119, 8992 –8995.
  47. 47. Magnetic nanoparticles The application of magnetic nanoparticles in cancer therapy is one of the most successful biomedical exploitations of nanotechnology. The efficacy of the particles in the treatment depends upon the specific targeting capacity of the nanoparticles to the cancer cells. Efficient, surface-engineered magnetic nanoparticles open up new possibilities for their therapeutic potential.… effective conjugation of folic acid on the surface ofsuperparamagnetic iron oxide nanoparticles (SPION) enables theirhigh intracellular uptake by cancer cells.Such magnetic-folate conjugate nanoparticles are stable for a longtime over a wide biological pH range: additionally, such particlesshow remarkably low phagocytosis as verified with peritonealmacrophages.
  48. 48. Conclusions• Nanomagnets can be made bioavailable by liposomal encapsulation with targeting• Nanoparticles smaller than 20 nm can be useful for local heat generation• Intracellular hyperthermia kills the cancer cell and releases heat shock proteins. These are used to target and kill other cancer cells.• Results in reduction in growth of tumor size• Nanomagnets can be used for MR Imaging in vivo 57
  49. 49. MICROBUBBLES• Used with ultrasound echocardiography and magnetic resonance imaging (MRI)• Diagnostic imaging - Traces blood flow and outlines images• Drug Delivery and Cancer Therapy
  50. 50. • Small (1-7 m) bubbles of air (CO2, Helium) or high molecular weight gases (perfluorocarbon).• Enveloped by a shell (proteins, fatty acid esters).• Exist - For a limited time only! 4 minutes-24 hours; gases diffuse into liquid medium after use.• Size varies according to Ideal Gas Law (PV=nRT) and thickness of shell.
  51. 51. ultrasound• Ultrasound uses high frequency sound waves to image internal structures• The wave reflects off different density liquids and tissues at different rates and magnitudes• It is harmless, but not very accurate
  52. 52. Ultrasound and Microbubbles• Air in microbubbles in the blood stream have almost 0 density and have a distinct reflection in ultrasound• The bubbles must be able to fit through all capillaries and remain stable
  53. 53. ShellAir or High MolecularWeight Gases 1- m
  54. 54. Preparation of microbubbles 1. Water 2. Fluorinated hydrocarbon 3. Polymer solution 4. Ethanol palmitic acid solution with Epikuron® 200 5. Homogeneization for 10 min at 12000 rpm
  55. 55. O2 microbubbles coated with PAAs Cationic PAA PAA-cholesterol Diameter = 549.5 ± 94.7 nm Diameter = 491.4 ± 38.2 nm PZ = 8.54±1.21 PZ = 6.22±1.17 pH = 3.28 pH = 6.50
  56. 56. Application of microbubble technology for ultrasoundimaging of the heart