Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

7 imaging


Published on

Published in: Business, Technology
  • Be the first to comment

  • Be the first to like this

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 (surface passivation) Hydrophobic crystals
  4. 4. What are quantum dots • Unique Spectral properties Broad absorption Narrow emission Wavelength depends on size CdSe/ZnS QD samples Annu. Rev. Anal. Chem. 2013. 6:143–62, Michalet , X. et al. Science. 2005, 307 , 538 – 44
  5. 5. Making hydrophobic quantum dots biocompatible • Various methods for making them watersoluble – Derivatizing surface with bifunctional ligands – Encapsulating in phospholipid micelles or liposomes – Polymer coating Gao , X. et al. Adv.Experim.Med. Biol. 2007, 620,57-73
  6. 6. Conjugating quantum dots to biomolecules • Avidin or protein-G with positively charged tail conjugated to negatively charged DHLA coat of quantum dots Goldman E.R. et al.( 2002 ) JACS,124 , 6378 – 82 ; Analytical Chemistry , 74 , 841 – 7 . Avidin protein G
  7. 7. 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
  8. 8. Quantum dots for multiplexing
  9. 9. Quantum dots for multiplexing benign prostate gland Multiplex Immunostaining gland with a single malignant cell Malignant HRS cells To differentiate Hodgkin’s from non-Hodgkin’s lymphoma B cells T cells Liu J et al. Anal. Chem. 2010, 82, 6237–6243; Am. Chem. Soc. Nano 4:2755–65
  10. 10. Quantum dots v/s other fluorescent probes Photostability (quantum dots do not photobleach) 3T3 cells Scale bar 10 µm. Red: qdot 605 Conjugate Green: Alexa488 Conjugate Wu et al. Nat. Biotechnol. 2003;21(1):41-62
  11. 11. Quantum dots v/s other fluorescent probes • Brighter than other fluorophores Quantum dots Fluorescein Larson et al. 2003
  12. 12. Quantum dots and imaging In vivo visualization of capillaries Quantum dots FITC-Dextran Larson et al. 2003, Science 300:1434-1436
  13. 13. Quantum dots and imaging anti-α-tubulin antibody anti-Her2 antibody Cancer cell surface marker red & green Microfilaments biotinylated phalloidin Actin filaments Nuclear antigens Wu et al. Nat. Biotechnol. 2003;21(1):41-62
  14. 14. Quantum dots and imaging Glycine Receptors Diffusion of single Qdot-GlyRs in synaptic boutons Primary antibody ↔ secondary Antibody – biotin ↔ QD Streptavidin Dahan et al. Science. 2003, 302:442-445
  15. 15. Quantum dots and imaging EGF receptor EGF-QD Live imaging of receptor mediated endocytosis Lidke et al. Nat. Biotechnol. 2004, 22:198
  16. 16. Quantum dots and imaging 1 µm 200 nm 200 nm Single quantum dot crystals can be observed in electron micrographs
  17. 17. Quantum dots and imaging: FRET
  18. 18. Quantum dots and imaging: FRET • Quantum dots have been used in FRET • In conjunction with Texas Red • In conjunction with fluorescent quenchers Willard et al. Nat Materials. 2003, 2:575
  19. 19. QDOTS IN VIVO
  20. 20. 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
  21. 21. 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
  22. 22. Imaging in vivo
  23. 23. Multifunctional NP for Imaging 24
  24. 24. Imaging techniques and related contrast agents X-ray  Iodinated contrast materials Au MRI  Gadolinium-based Fe-based 19 F-based PET  Radioactively labelled agents (11C, 18F)
  25. 25. X-ray CT CT is ubiquitous in the clinical setting as. The increasing use and development of micro-CT and hybrid systems that with PET, MRI. The most investigated NPs in this field are gold NPs, since they have large absorption coefficients against the x-ray source used for CT imaging and may increase the signal-to-noise ratio of the technique. To date, different types of gold NPs have been tested in a preclinical setting as contrast agents for molecular imaging: nanospheres, nanocages, nanorods and nanoshells. Gold NPs formulations as an injectable imaging agent have been utilized to study the distribution in rodent brain ex vivo
  26. 26. nanocage nanorod nanoshell nanosphere Size 4-40 nm
  27. 27. PET The strategy utilized is consisting in incorporating PET emitters within the components of the NP, or entrapping them within the core. brain cancer Oku et al (2011), Int. J. Pharm. 403 :170–177
  28. 28. MRI MRI relies upon the enhancement of local water proton relaxation in the presence of a contrast agent (CA). CA are compounds that catalytically shorten the relaxation times of bulk water protons. T1 (longitudinal relaxation– in simple terms, the time taken for the protons to realign with the external magnetic field) Positive CA (Gd) T2 (transverse relaxation –in simple terms, the time taken for the protons to exchange energy with other nuclei) Negative CA (Iron oxide agents )
  29. 29. Gd-based NP several nanotechnological approaches have been devised, based on 2 ideas: -carrying many Gd chelates; -slow down the rotation of the complex Examples include liposomes micelles dendrimers fullerenes. However, this approach has not yet achieved clinical applications.
  30. 30. Magnetic Nanoparticles magnetic NPs (MNPs) are of considerable interest because they may behave either as contrast agents or carriers for drug delivery. Among these, the most promising and developed NP system is represented by superparamagnetic iron oxide agents 31
  31. 31. 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 . • 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. B 32
  32. 32. The most promising and developed NP system is represented by superparamagnetic iron oxide agents, consisting of a magnetite (Fe3O4) and/or maghemite (Fe2O3) crystalline core surrounded by a low molecular weight carbohydrate (usually dextran or carboxydextran) or polymer coat. Iron oxide NPs can be classified according to their core structure, such as Monocrystalline (MION; 10–30 nm diameter), or according to their size as ultra-small superparamagnetic (USPIO) (20–50 nm diameter), superparamagnetic (SPIO) (60–250 nm).
  33. 33. J Lodhia et al. Biomed Imaging Interv J 2010; 6(2):e12
  34. 34. Polymer Coated Magnetite Nanoparticles Dextran, silane, poly-lactic acid, PEG, dextran, chitosan, gelatin, ethylcellulose…
  35. 35. Formation of Nanoparticles • Solution of Dextran, FeCl3.6H2O and FeCl2.4H2O (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 36
  36. 36. Variation of Formation • Change Coating Material • Crosslinking coating material – Increases plasma half-life – Same Particle Size Lee H et al. J. AM. CHEM. SOC. 2007,129, 1273-12745
  37. 37. Magnetite Cationic Liposomes (MCL) Fe3O4 • Why Cationic? – Interaction between + liposome and – cell – membrane results in 10x uptake.
  38. 38. Formation of MCL • magnetite NP dispersed in distilled water • N-(α-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 DLPC DOPE
  39. 39. 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
  40. 40. 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. Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328 42
  41. 41. 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 Field Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328 43
  42. 42. Effect of Hyperthermia Treated Tumor Before Treatment Untreated Tumor Rectum antitumor immune response After Treatment
  43. 43. 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.
  44. 44. 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 46
  45. 45. Magnetic Nanoparticles/Carriers M • Magnetite Core • Starch Polymer Coating M M M • Bioavailable • Phosphate in coating for functionalization • Chemo Drug attached to Coating M Magnetite Core Starch Polymer M M • Mitoxantrone • Drug Delivered to Rabbit with Carcinoma 47
  46. 46. Results of Drug Delivery • External magnetic field (dark) • deliver more nanoparticles to tumor • No magnetic field (white) • most nanoparticles in non tumor regions Alexiou C et al ANTICANCER RES 27: 2019-2022 (2007) 48
  47. 47. Magnetic nanoparticles in medicine They consist of a metal or metallic oxide core, encapsulated in an inorganic or a polymeric coating, that renders the particles biocompatible, stable, and may serve as a support for biomolecules. • Drug or therapeutic radionuclide is bound to a magnetic NP, introduced in the body, and then concentrated in the target area by means of a magnetic field. • Depending on the application, the particles release the drug or give rise to a local effect (hyperthermia). • Drug release can proceed by simple diffusion or take place through mechanisms requiring enzymatic activity or changes in physiological conditions (pH, osmolality, temperature, etc…).
  48. 48. 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 50 Yang, etal. Angew. Chem. 2007, 119, 8992 –8995.
  49. 49. 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 of superparamagnetic iron oxide nanoparticles (SPION) enables their high intracellular uptake by cancer cells. Such magnetic-folate conjugate nanoparticles are stable for a long time over a wide biological pH range: additionally, such particles show remarkably low phagocytosis as verified with peritoneal macrophages.
  50. 50. 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 52 vivo
  51. 51. MICROBUBBLES • Used with ultrasound echocardiography and magnetic resonance imaging (MRI) • Diagnostic imaging - Traces blood flow and outlines images • Drug Delivery and Cancer Therapy
  52. 52. MICROBUBBLES • Small (1-7 µm) 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.
  53. 53. 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
  54. 54. 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 Soft Matter, 2008, 4, 2350–2359
  55. 55. Shell Air or High Molecular Weight Gases 1-7µm
  56. 56. Preparation of microbubbles - Homogeneization - Sonication - microfluidic T-devices
  57. 57. O2 microbubbles coated with PAAs Cationic PAA Diameter = 549.5 ± 94.7 nm PZ = 8.54±1.21 pH = 3.28 PAA-cholesterol Diameter = 491.4 ± 38.2 nm PZ = 6.22±1.17 pH = 6.50
  58. 58. Application of microbubble technology for ultrasound imaging of the heart