1. Molecular Imaging Module A Dipanjan Pan, PhD, FRSC Department of Bioengineering Beckman Institute of Advanced Imaging and Science Department of Materials Science and Engineering dipanjan@illinois.edu

2. Topics to cover: Module A: Basics of imaging Module B: Biological Barrier Module C: Molecular Imaging with Clinical modalities Module C: Molecular Imaging with pre-Clinical modalities Optional Book: Nanomedicine (ed. By Dipanjan Pan, CRC Press) 1 Midterm Quiz, 2 HW, 1 Final

3. The First Image Ever Taken http://www.hrc.utexas.edu/exhibitions/permanent/firstphotograph/ View from the Window at Le Gras, Joseph Nicéphore Niépce circa 1826 1838 Taken by Louis Daguerre, in Paris http://www.burnstudio.co.za/blog/first-photo-of-a-human-being/

4. How It All Started? U.S. President James Garfield was shot in 1881 Physicians were unable to determine if the bullet had entered a vital organ. This knowledge was needed quickly to decide the medical treatment required to save Garfield’s life. For the next 80 days, 16 doctors were consulted. The first doctor, stuck a non-sterile finger into the wound followed this by inserting a non sterile probing instrument to find the bullet. He never found the bullet. Followed by the navy surgeon general who searched with his finger so deeply that he really did puncture the liver. President James Garfield 20th President of the US Mar 4, 1881-Sept 19, 1881 Alexander Graham Bell Inventor of phone Mar 3, 1847 – Aug 2, 1922

5. Alexander Graham Bell had just invented telephone and an induction coil (metal detector) Alexander Graham Bell rigged up a crude metal detector to help find the bullet. Bell had located the bullet and it was much deeper than was originally thought. With Garfield's condition growing steadily worse, doctors decided to cut him open to remove the bullet. It was not found. What Bell had actually located so deep in the body was the metal spring under the mattress!! Failed but “first” attempt to locate something inside human body Noninvasively

6. Why We Need Imaging for Human Health? Various imaging methods cut through darkness noninvasively Fluorescence, ultrasound, computed tomography (CT), positron emission tomography, Magnetic resonance imaging Because it is dark inside! Bioluminescent Jellyfish

7. 1895 The first x-ray of the human body, taken by Conrad Röntgen on November 8, 1895. He called the new form of radiation he had discovered x-rays, with the X standing for unknown. Image of Röntgen’s wife's hand The first X-ray image ever recorded 116 yrs Sharper X-ray image First Medical Image Ever Taken

8. An x-ray revealed that an 18-month-old boy had swallowed two disc batteries. (Source: Cedars-Sinai)

9. This CAT scan shows calcification in the bilateral carotid, bilateral subclavian, and brachiocephalic veins of Hatiay, a male Egyptian scribe aged 40–50 yrs, (1570–1293 Before Common Era), Probing the Past

10. CT image of a broken arm with pins CT image of a hip replacement

11. http://www.cedars-sinai.edu/Patients/Programs-and-Services/Imaging-Center/Image-Gallery-images/CapAngio01-Color-Pirouette.gif A volume rendering (VR) of a three-dimensional set of computed tomography (CT) images shown as a two-dimensional projection. Aortic aneurysm

12. Heart valve, left, and heart valve in a heart, right. left: Normal subclavians; right: abnormal subclavians with positive occlusion (arrow).

13. A specialized MRI of the Brain with Diffusion Tensor Imaging (DTI) Analysis showing the neural pathways, which displays how different parts of the brain are connected. When the pathways are tangled it can be an indication of shaken-baby syndrome.

14. “A man’s brain being removed and cut into hundreds of pieces to be handed out all around the world—without his family’s knowledge……..”

15. Year 1955: Autopsy on Albert Einstein was performed by pathologist Thomas S. Harvey Photographed the scientist’s brain Shared slices of it with fellow pathologists! Harvey kept pictures of the dissected brain and pieces (170) of it for himself! No permission for the removal and preservation had been given by Einstein or his family Year 2007: Harvey died Items eventually unearthed and given to the National Museum of Health & Medicine, in Silver Spring, Md. The Story Harvey with Einstein’s ‘pickled’ brain

16. MRI Distribution Maps of Corpus Callosum Thickness Between Einstein and the Old Age Control Group Brain2013, DOI: 10.1093/brain/awt252).10.1093/brain/awt252

17. MRI Distribution Maps of Corpus Callosum Thickness Between Einstein and the Young Age Control Group

18. Measurements of corpus callosum (CC) morphology and brain between Einstein and the two different age control groups

19. Close to Our Heart Paul C Lauterbur (May 6, 1929 -Mar 27, 2007) Sir Peter Mansfield Nobel Prize for MRI, 2003 When Lauterbur’s paper was rejected by Nature, however, after his persistance, it was published and is now acknowledged as a classic Nature paper. The Nature editors pointed out that the pictures accompanying the paper were too fuzzy They were the first images to show the difference between heavy water and ordinary water! Nature 242, 190-191 (1973) Raymond Damadian, First MRI Patent, 1974

20. Why Contrast Agents?

22. Types of Contrast Agents-1 Modality Specific Radio Contrast Agent Improve the visibility of internal structures in X-ray based imaging Cerebral angiogram MRI Contrast Agent altering the magnetic properties of nearby hydrogen nuclei Stroke Sonographic Contrast Agent Sound waves are reflected from interfaces between substances (backscattering) Hepatocellular carcinoma Nuclear Contrast Agent Whole body PET scans showing the distribution of radio-labeled monamine oxidase Optical Contrast Agent A mouse’s brain cerebellum tumor in optical imaging

23. Types of Contrast Agents-2 Biological Targeting

24. Orange areas of a PET/CT image indicate the uptake of 18F-fluoro-2-deoxy-D-glucose in a primary cancer lesion and a lymph node (Sam Gambhir, Stanford) Colon cancer scan captured by GE's PET/CT and the imaging agent FDG. The fused volume rendering of a PET/CT angiography provides both vascular and metabolic information. SEEING BLOOD A normal mouse reveals its vasculature with Fenestra. Bone (gray), vessels (red), and skin (brown)

25. Trends in Imaging En route to ‘Molecular’ Imaging Anatomical Imaging Physiological Imaging Molecular Imaging StructureMechanismTarget Morphology Morphometry Hemodynamics Vascular Permeability Tissue oxygenation/hypoxia CNS activity Metabolites pH Receptor mediated imaging Targeted contrast agents In vivo distributive properties

26. Personalized Medicine Changing the Healthcare Clinical Symptoms Screening Early Diagnosis Preventive Treatment Functional and quantitative imaging localizing the disease Follow the disease response to therapy Minimally invasive treatment management tailored to the patient

27. A Paradigm Shift in Healthcare Clinical Symptoms Screening Early Diagnosis Preventive Treatment Image source: wiki Challenge? Understanding root causes of disease Linking symptoms to molecular origins Bring bench side science to clinics Pan et. al. Eur J Radiol. 2009, 70(2):274-85. Pan et. al. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010 Sep 21.

28. Comparison of resolution and imaging depths of the modalities; the "pendulum" length represents imaging depth, and the "sphere" size represents resolution. Modified from http://obel.ee.uwa.edu.au/research/oct/intro/ So Many Modalities “Speckle artifacts” Slow; Strong influence from motion Fast Photoacoustic tomography Spectral Computed tomography Quantitative Ca interference Things to consider Resolution Depth penetration Speed of acquisition Quantification Sensitivity Ca interference No single modality is ideal for every application BioE 498/598DP

29. Resolution and Contrast Material Concentration

30. What Structure You Would Like to Resolve?

32. Targeting Approaches There is a search dual-mode probes that can detect a tumor imaging) and destroy it (therapy) Based on retention effect of particle of certain hydrodynamic size in cancerous tissues (e.g. Doxil) DRUG PROBE DRUG PROBE Physical Targeting Active Targeting Passive Targeting Based on nanoparticle functionalization for specific targeting of disease cells DRUG PROBE pH-sensitive Temperature Redox-potential Ultrasound Proteins (antibodies and their fragments such as TAT), nucleic acids (aptamers), receptor ligands (peptides, vitamins, and carbohydrates

33. Site specific delivery- active or passive targeting RES Lungs Liver Spleen Reticuloendothelial System (RES) Site Specific Delivery

34. Even with controlled delivery side effects cannot be avoided Might cause severe side effects- spread over body Site specific delivery- active or passive targeting

35. Enhanced permeability and retention (EPR) effect Disorganization of tumor vasculature Poor lymphatic drainage Passive Targeting of Particulate by EPR Mechanism

36. Ringsdorf’s Model Active Targeting

37. Patho-physiology of Tumor Tissue Angiogenesis Hypervasculature Impaired lymphatic drainage ***Due to these characteristics, tumors can be exploited for tumor-selective accumulation****

38. 38 Angiogenesis Biological process of forming new blood vessels

39. Enhanced permeability and retention (EPR) effect Disorganization of tumor vasculature Poor lymphatic drainage Passive Targeting of Particulate by EPR Mechanism

40. Module B Biological Barriers

41. DRUG PROBE DRUG PROBE Biological Barriers External barriers En route barriers Cellular barriers SkinMucosa BloodExtracellular matrix Endosomal/lysosomal degradation Inefficient translocation to the targeted sub-cellular organelles Cellular Delivery Human barriers

42. Common Routes of Administration

43. Human Barrier (Errors)

44. First Pass Mechanism Metabolism occurs during the absorption process. The fraction of the initial dose appearing in the portal vein is the fraction absorbed, and the fraction reaching the blood circulation after the first-pass through the liver defines the bioavailability of the drug.

45. Source: Grays Anatomy Histologic image of human epidermis

46. Source: DOI: 10.5772/23951 Composition of gastric mucus

48. Possible destabilization and degradation pathways of probes during in vivo circulation Immunoglobulins, complement proteins, albumin, apolipoprotein and fibrinogen. adsorbs on the surface of nanoparticles and tag them for attack by the MPS. Scavengers to engulf foreign particles Mononuclear phagocyte system: (MPS)

49. Renal Clearance Renal molecular weight cut-off: 48kDa Renal size cut-off: ~10 nm Anything beyond >10-20 nm may not be excreted Size: ~10 nm

50. Blood Brain Barrier (BBB) Blood and brain junction, endothelial cells are tightly stitched together Composed of smaller subunits, e.g. biochemical dimers, transmembrane proteins, occludin, claudins, junctional adhesion molecule (JAM), ZO-1 protein Crossing BBB: disruption by osmotic means; biochemically by the use of vasoactive substances such as bradykinin; localized exposure to high-intensity focused ultrasound (HIFU) Pore size upper limit ~12 nm (malignant glioma) Polyethylenglycol, peptides….. A cortical microvessel stained for blood-brain barrier protein ZO-1

51. Cellular Barriers Possible degradation routes Acidic pH and enzymes (late endosomes -lysosomes). Viscosity and intracellular enzymes of the cytosol. Recycling (exocytosis) of the vesicle contents. Excretion Degraded nanoparticle SUCCESS FALIURE

52. Gases Hydrophobic molecule Polar (large) Polar (small) Charged molecule Diffusion of Agents Through Cellular Bilayer Hydrophobic molecule Charged molecule Polar (large) Glucose Polar (small) H 2 O, ethanol (a) ibuprofen, (b) aspirin, (c) erythromycin Charged molecule: activity of specific transport and channel proteins

53. Direct translocation across the plasma membrane is another suggested endocytic pathway Does not depend on the metabolic activity of the cells. Energy-independent Receptor-independent Transduction Cell penetration peptides Can There be a Direct Access to the Cytoplasm? How can we avoid endosomal escape pathway?

54. 1988 1994 1996 1997 2000 2001 2004 2006 2008 Discovery TAT Covalent approach PENETRATIN TRANSPORTAN Complex approach MPG POLY R TP10 In vivo PEP-1 Clinical Trial PPTG SAP SynB M918 PrPr EB1 CADY Phase IIb-3 Cell Penetrating Peptides (CPPs)

55. Extra Vascular NP: How Far Below We Could Drive the Size Down? Pan, Turner, Wooley Macromolecules, 2004, 37 (19), pp 7109–7115

56. Its all about ‘CONTROL’

57. Precise Targeting (Tissue/Cell/Molecular) Precise Action (Maximize therapeutic action and minimize toxicity and side effects) Precise Timing (On when it is needed, Off when it is not needed) Implicit in these design goals is the requirement for precise control mechanisms that can either respond to local environments automatically or respond to signals sent remotely. A perfect Approach

58. (1) Increase localization in the tumor through: (a) Passive targeting (b) Active targeting (2) Decrease localization in sensitive, non-target tissues (3) Ensure drug leakage during transit to target (4) Protect the agent from degradation and from premature clearance (5) Retain the agent at the target site for the desired period of time (6) Facilitate cellular uptake and intracellular trafficking (7) Biocompatible and biodegradable Characteristics of an ideal tumor-targeted Agent Lammers T, et al. British Journal of Cancer 2008;99:392-397.

59. Absorption, Distribution, Metabolism, and Excretion (ADME) Describes the disposition of a pharmaceutical compound within an organism. The four criteria all influence the drug levels and kinetics of drug exposure to the tissues. Influence the performance and pharmacological activity of the compound as a drug. LADME: L stands for "liberation" and deals with details of the route of administration such as what a tablet will do at a given gastric pH level, the creation of extended-release injectables for IM or SC use etc.

60. Absorption Metabolism Liver Other RES sites Distribution Target tissue Excretion Kidney Systemic Circulation Local barriers ADME

61. Clinically Utilized Drug Targeting Strategies

62. EPR: Taking advantage of retention A. Tumorous tissues suffer of Enhanced Permeability and Retention effect (RES) B. Nanoparticles injected in the blood stream do not permeate through healthy tissues C. Blood vessels in the surrounding of tumorous tissues are defective and porous D. Nanoparticles injected in the blood permeate through blood vessels toward tumorous tissues, wherein they accumulate Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88

63. Clinical Example of EPR Doxil is a polyethylene glycol coated liposomal formulation of doxorubicin. Marketed by Ben Venue Laboratories of J&J. Outside the US, Doxil is known as Caelyx (Janssen). Approved by the FDA for treatment of ovarian cancer and multiple myeloma and an AIDS-related cancer.