1. Ultrasound Imaging (Contrast Agents and Therapy) MEng in Bioinstrumentation

2. Why Ultrasound? Over half a century old technique! Arguably the most widely used imaging technologies in medicine. Portable, free of radiation risk, and relatively inexpensive compared to MRI, CT and PET Tomographic, i.e., offering a “cross-sectional” view of anatomical structures. “Real time,”- providing visual guidance for interventional procedures

3. Do you expect any similarities?

4. Most amazing is that sound can actually help us to see what is hidden, just like the way bats 'see'. Bats always have the night shift. They go hunting for things to eat at night where food isn't well lit. Fortunately, bats are gifted with a system of locating things with sound. First they emit sound.

5. The human ear cannot hear below 20 Hz. Elephants can use infra sound. The human ear cannot hear above 20,000 Hz. Bats use ultrasound to locate food. Dolphins use it to communicate. Ultrasound used in medical imaging operate at frequencies way above human hearing: about 2 million Hz - 20 million Hz (2-20 MHz).

6. Sound travels in waves. Ultrasound physics has to do with the higher frequencies of sound. Human hearing is from about 20 cycles per second or 20HZ (a low hum) to about 20,000 cycles per second or 20KHZ. A grasshopper sends out sound waves at 40KHZ. A dog can hear at about 30KHZ and bats send chirps and listens for the echoes at 100KHZ.

7. Properties of Sound Waves The number of cycles occurring in one sec of time (cycles per sec) The high frequency wave sounds higher than the low freq wave High Frequency Wave Period Time Pressure Low Frequency Wave Period Time Pressure http://www.genesis-ultrasound.com/Ultrasound-physics-2.html wavelength Crest Trough Amplitude Frequency Velocity Wavelength Amplitude Units to describe frequency: Hertz= 1 cycle in one sec kHz= 1000 Hz= 1000 cycles per sec MHz= 1000000 Hertz US imaging frequency range: 2-12 MHz

8. wavelength Wavelength Length of space over which one cycle occurs (distance) wavelength Distance Given a constant velocity, as frequency increases wavelength decreases (V= x f) Common US frequencies and wavelengths -2.25MHz = 0.6 microns -5.0 MHz = 0.31 microns -10.0 MHz = 0.15 microns

9. High frequency US waves  High axial resolution  More attenuation  Superficial structure Ultrasound Wavelength and Frequency Low frequency US waves  Lower resolution  Less degree attenuation  Deeper penetration Higher frequency waves are more highly attenuated than lower frequency waves at a given distance High frequency transducers (10-15 MHz) to image superficial structures (e.g. stellate ganglion blocks) Low frequency transducers (2-5 MHz) to image the lumbar neuraxial structure

10. Velocity Average speed of US in the human body is 1540 m/sec Directly related to the stiffness of media Inversely related to the density of media Slowest in air/gasses fastest in solids Medium Velocity (m/sec) -------------------------------------------- Air 330 Fat1450 Water1480 Soft tissue1540 Blood1570 Muscle1580 Bone4080 c = × f = c / f

11. Amplitude The strength/intensity of the sound wave at any given point in time Represented by the height of the wave Amplitude/intensity decreases with increasing depth Magnitude of the pressure changes along the sound wave Power: rate at which energy is transferred from a sound beam- proportional to the amplitude squared Intensity (Watts/cm 2 ) is the concentration of energy in a sound beam

12. The ultrasound amplitude decreases in certain media as a function of ultrasound frequency (attenuation coefficient) ScN-Sciatic nerve, PA - Popliteal artery. 8 MHz 10MHz 12MHz Practical consequence of attenuation: the penetration decreases as frequency increases Attenuation Coefficient

13. Ultrasound frequency affects the resolution of the imaged object. Resolution can be improved by increasing frequency and reducing the beam width by focusing. 8 MHz 10MHz 12MHz For a constant acoustic velocity, higher frequency US can detect smaller objects and provide a better resolution image. A 0.5-mm- diameter object

14. Spatial Resolution Axial and Lateral. Axial resolution is the minimum separation of above-below planes along the beam axis. It is determined by spatial pulse length, which is equal to the product of wavelength and the number of cycles within a pulse. Axial resolution = wavelength (λ) × number of cycle per pulse (n) ÷ 2

15. Common Frequencies for Clinical US Dystrophic calcification of the choroids Portal Vein Ultrasound Color Doppler imaging shows a thrombus in upper PV moderately dilated (14.5 mm) with splenomegaly: Cirrhosis with PV thrombosis. MRI of a large tumor in the left kidney (L) and 12 days following HIFU treatment (R). Ablative therapy

16. T1: ultrasonic generator, Q1: transmitter, Q2: receiver, T2: converter amplifier, W: water bath, L: light, P: photographic/ heat-sensitive paper Ultrasound in Med. & Biol., Vol. 30, No. 12, pp. 1565 - 1644, 2004 Dr. Karl Theo Dussik, an Austrian neurologist, was the first to apply US to image the brain.

18. High echogenicity Low attenuation Low blood solubility Low diffusivity Ability to traverse pulmonary system Lack of biological effects in repeat exposures Ideal Characteristics of an Ultrasound Probe

19. Contrast Agents and Their Distribution Relative to the Vascular Endothelial Cells

20. Diagram illustrating development stage of microbubbles, nanobubbles, and nanodroplets for diagnostic and therapeutic purposes. HIFU = high-intensity focused ultrasound; KDR = kinase domain receptor.

21. Lindner JR. Microbubles in medical imaging: current applications and future directions. Nat Rev Drug Discov. 2004 Jun;3(6):527-32. Microbubbles Response to US Microbubbles volumetrically oscillate due to continuous changes in the external pressure caused by an acoustic sound wave. At high pressure amplitudes, microbubbles collapse violently, producing water jetting and shock waves, and other inertial phenomena.

22. A slow motion capture of interial cavitations of micro bubbles Right frame: Image using both tissue and contrast agent fundamental frequency backscatter Left frame: the fundamental tissue signal has been filtered leaving the higher harmonic signal corresponding only to the contrast agent and, subsequently, the blood flow of the liver. Contrast harmonic image of liver in vivo

23. Lindner JR. Microbubles in medical imaging: current applications and future directions. Nat Rev Drug Discov. 2004 Jun;3(6):527-32. Acoustic Properties of Microbubbles

24. Non Linear Response of the Microbubble

26. Microbubble Contrast Agents NameManufacturerShellGasMean Size (µm) AlbunexMolecular Biosystems AlbuminAir4.3 OptisonMallinckrodt/ Amersham AlbuminOctafluoropropane2-4.5 DefinityBristol-Myers Squibb Lipid/surfactantOctafluoropropane1.1-3.3 ImagentImacorLipid/surfactantN 2 / perfluorohexane 6.0 SonovueBracco Diagnostics LipidSulphur hexafluoride2-3 LevovistSchering AGLipid/GalactoseAir2-4

27. Bubbles coalescing in the extravascular space become detectable by US due to their micron sized collections Orthotopically implanted human breast adenocarcinoma xenograft (MDA-MB-231 cells) in nude mouse imaged following intravenous administration of 5×10 7 VEGFR2- targeted probe. Scale bar =3 mm Ultrasonic Molecular Imaging of Tumor Angiogenesis in Breast Cancer

28. Albunex Air-filled sonicated albumin MBs (MBI, San Diego, California) 1994: First ultrasound CA approved for use in US Albumin shell designed to prevent outward diffusion of air from MBs Substantial loss of gas volume occurred during transit to systemic circulation following intravenous injection Markedly decreased contrast enhancement and short duration of clinically useful contrast

29. Newer Agents Newer agents designed to improve intravascular stability Modifications in shell and gas content “Air-tight” polymer shells or lipid-galactose stabilized shells designed to minimize outward diffusion of gas Use of gases less prone to outward diffusion than air Inert high-molecular-mass gases with low diffusion coefficients and low solubility in water (low Ostwald coefficient); result in prolonged lifespan of MBs

30. Optison TM Perflutren Protein-Type A Injectable Microspheres GE Healthcare, Buckinhamshire, UK Octafluoropropane Manufacturer has voluntarily suspended marketing since 2005 http://www.amershamhealth-us.com/optison/monograph/om03-02.html Optison with RBCs Structural Formula

31. Definity FDA approval in 2001 Bristol-Myers Squibb Medical Imaging, Billerica, MA $65 million in sales in 2006 More than 2 million patients dosed

32. How Do We Make These Bubbles?

33. SEM photograph of MBs (50 000×)

34. Subcutaneous human ovarian adenocarcinoma xenograft tumor (arrows) imaged with targeted contrast-enhanced ultrasound imaging after intravenous injection of integrin-targeted (A) and MB- (control) (B). Imaging signal obtained after MB-targeted injection was substantially higher than that obtained after control injection.

35. The prepared nanoparticles in vitro released the drug steadily for 72 hours without occurrence of burst release. Docetaxel MBs Loaded with Chemotherapeutics

36. Influence of ultrasound exposure on tumor temperature After ultrasound exposure, the tumor temperature increased steadily in a time dependent manner. The growth curve of hepatocellular carcinoma xenografts.The tumor volume increased persistently in model group, while in all the docetaxel treated groups, the tumor volume increment was inhibited, especially in the GDN+UE group.

37. Bubble diameter (2r) (µm) (Pa) (atm) 10002880.00284 3.0960000.947 0.39600009.474 The Laplace pressure is the pressure difference between the inside and the outside of a curved surface. The pressure difference is caused by the surface tension of the interface between liquid and gas. The Laplace pressure is determined from the Young–Laplace equation given as where R1 and R2 are the radii of curvature and  is the surface tension. Laplace Pressure

38. Phase-change contrast agents (PCCAs) Consist of perfluorocarbons (PFCs) that are initially in liquid form, but can then be vaporized with acoustic energy. Exposing pre-formed PFC micro bubbles to decreased ambient temperature and increased ambient pressure results in condensation of the gaseous core. The decreased size results in an increased Laplace pressure, which serves to preserve the particle in the

39. Phase-change contrast agents (PCCAs) Exposing pre-formed PFC micro bubbles to decreased ambient temperature and increased ambient pressure results in condensation of the gaseous core. The decreased size results in an increased Laplace pressure, which serves to preserve the particle in the PCCAs take advantage of the volumetric increase that occurs during vaporization. Microscale droplets limited to flow in the vascular space have been proposed for a number of techniques, including temporary occlusion of blood vessels and enhancing cavitation activity. PCCAs manufactured with diameters in the 100-nm range may be able to diffuse out by EPR before phase-transition Contrast-enhanced extravascular imaging, cell-specific targeting, drug delivery, and therapy via ultrasound.

40. A microscale droplet of MBs exposed to a 0.25 µs pulse at approximately 0.55 MPa vaporizes to form a gas bubble approximately 5-fold larger

41. Bright field (a, c) and fluorescence (b, d) microscopy illustrating that the shell is preserved when DiI-labeled DFB microbubbles (a, b) are condensed to the liquid state (c, d), the shell is preserved through the change in volume.

42. Schematic illustration of drug transfer from nanodroplets through microbubbles into cells under the action of ultrasound. PFP/PEG-PLLA microbubbles in a plasma clot before (A) and after sonication for 1 min by 1- MHz, 3.4 W/cm 2 (B) and 90-kHz, 2.8 W/cm 2 ultrasound (C) at room temperature. Therapy with PCCA

43. Fig. 2AFig. 2A. PEG-PCL nanodroplets inserted in PBS through a 18 G needle (left) or 26 G needle (right). Bubbles formed when nanoemulsion is injected through a thin needle are visualized as bright spots (indicated by arrows in the right panel); bubbles rise to the sample surface while droplets precipitate to the bottom of a test tube. Fig. 2BFig. 2B. PEG-PCL nanodroplets injected in the agarose gel through a 18 G (left) or 26 G (right) needle. Injection through the thin needle results in immediate formation of very bright bubbles whose size and brightness increase with time; the brightness of the droplets also increases with time suggesting a post-injection droplet-to-bubble transition.

44. Photographs of a mouse bearing two ovarian carcinoma tumors (A) - immediately before and (B) - three weeks after the treatment; a mouse was treated by four systemic injections of nanodroplet-encapsulated PTX, nbGEN (20 mg/kg as PTX) given twice weekly; the right tumor was sonicated by 1-MHz CW ultrasound (nominal output power density 3.4 W/cm 2, exposure duration 1 min) delivered 4 hours after the injection of the drug formulation. Ultrasound was delivered through a water bag coupled to a transducer and mouse skin by Aquasonic coupling gel.

45. Effective regression of the ovarian carcinoma tumor treated by nbGEN/ultrasound combination as described in Materials and Methods. The first photograph was taken before the start of the treatment, the second – two weeks later, i.e. immediately after the last treatment of the first treatment round. The third photograph was taken one week after the completion of the first treatment round. Fig. 6BFig. 6B. Normalized tumor growth/regression curve for the mouse presented in Figure 6A.Figure 6A

46. Bubble-mediated Gene Delivery

47. US and Bubble-mediated Drug Delivery Through BBB

48. Ultrasound and pH Dually Responsive Polymer Vesicles for Anticancer Drug Delivery Upon ultrasound radiation, the disruption and re-self-assembly lead to smaller vesicle. Upon decreasing the solution pH, the shrinkage of vesicles by the recrystallization of PTMA chains surpasses the swelling of vesicles by the partial protonation of DEA, leading to smaller vesicles. Further decreasing the solution pH will lead to the complete protonation of DEA and finally the disassembly of vesicles. Both ultrasound radiation and decreasing pH can lead to faster drug release.

49. A) Recanalization was observed in angiograms taken post-HIFU treatment. 2/4 (50%) of animals treated with 415 W power, exhibited recanalization when compared to post- stroke angiograms. There was no damage or leakage from the vessel apparent on the angiogram. B) Post-treatment H&E showed no evidence of tissue damage or hemorrhage due to HIFU in the region of sonication or in the surrounding brain tissue. Scale bar = 500 µm. C) Prussian blue staining was used to detect small fragments of the disrupted iron- loaded blood clots. doi:10.1371/journal.pone.0042311.g003

50. Intravital fluorescence images of subcutaneous pancreatic tumors before and after FUS treatment

51. (A) and (B) – two coronal slices in the 19 F MR images superimposed on the low resolution proton anatomic images of a pancreatic tumor bearing mouse. A 2% PFCE nanoemulsion stabilized with PEG-PCL copolymer was systemically injected every two hours, 200 μl each. Four injections were given, for a total of the PFCE dose of 2 mmol/kg. Images were recorded an hour after the last nanodroplet injection (seven hours after the start). (C)- Multiple liver metastases (arrows) are revealed at the necropsy of the mouse (indicated by long thing arrows). Transparent large tumor is indicated by a thick arrow. Organs could be displaced at necropsy.

52. Dramatic regression of a breast cancer MDA MB231 tumor treated by four systemic injections of paclitaxel loaded 1% PFCE/0.25% PEG-PCL nanoemulsion and focused 1-MHz CW ultrasound applied for 60 s

53. FDA “Black Box” Warning Issued on October 10, 2007 Post-marketing reports of 11 deaths 1-12 hours following administration of perflutren-based contrast agents 10 patient deaths following Definity injection and 1 death following Optison injection 4 patient deaths temporally related to contrast injection Perflutren-based compounds contraindicated for use in patients with: 1. Acute coronary syndromes 2. Acute myocardial infarction 3. Worsening or clinically unstable heart failure http://www.fda.gov/cder/drug/InfoSheets/HCP/microbubbleHCP.htm

54. Safety Data Adverse Experience Placebo (n=42) 5 ul/kg (n=85) 10ul/kg (n=85) All Perflutren (n=169) Headache3 (7%)4 (5%)5 (6%)9 (5%) Dizziness1 (2%)2 (2%)1 (1%)3 (2%) Back Pain003 (4%)3 (2%) Nausea02 (2%)1 (1%)3 (2%) Flushing002 (2%)2 (1%) Chest pain002 (2%)2 (1%) Pruritis01 (1%) 2 (1%) Rash01 (1%) 2 (1%) Sweating01 (1%) 2 (1%) IV Site Pain1 (2%)1 (1%)0 Fatigue1 (2%)1 (1%)0 Kitzman DW, Goldman ME, Gilliam LD, Cohen JL, Aurigemma GP, Gottdiener JS. Efficacy and safety of the novel ultrasound contrast agent perflutren (definity) in patients with suboptimal baseline left ventricular echocardiographic images. Am J Cardiol. 2000 Sep 15;86(6):669-74.

55. Safety Data No clinically significant change in physical examination, vital signs, electrocardiographic tracings, or chemistry or hematology laboratory values Adverse event rates similar across treatment groups 30 of 169 patients (18%) in the combined perflutren-treated group (15% in the 5 ml/kg group and 20% in the 10 ml/kg group) 6 of 42 placebo-treated patients (14%) Headache was most frequently reported adverse event (9 of 169 patients who received perflutren (5%) and 3 of 42 patients who received placebo (7%) Kitzman DW, Goldman ME, Gilliam LD, Cohen JL, Aurigemma GP, Gottdiener JS. Efficacy and safety of the novel ultrasound contrast agent perflutren (definity) in patients with suboptimal baseline left ventricular echocardiographic images. Am J Cardiol. 2000 Sep 15;86(6):669-74.

56. “Pseudocomplication” Main ML, Goldman JH, and Grayburn PA. Thinking outside the “Box”—the ultrasound contrast controversy. J Am Coll Cardiol 2007; 50 (25): 2434-2437. Complications occurring after a medical procedure may be due to either the procedure itself or due to progression of the underlying disease state Major cardiovascular events are more likely to occur in patients who are “ill enough” to require diagnostic testing Echocardiography often the test of choice (or the only test available) for critically ill patients (shock, hypotension, tamponade, etc.) Association of adverse events following contrast administration does not establish causality