Ultrasound Targeted Microbubble Destruction Research Project

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This is a presentation of my final research project for my Introduction to Biomedical Engineering course.* Together with a partner, I conducted a thorough internet research of the newly emerging …

This is a presentation of my final research project for my Introduction to Biomedical Engineering course.* Together with a partner, I conducted a thorough internet research of the newly emerging field of drug delivery using microbubbles activated by ultrasound technology. We presented to Biomedical Engineering faculty, graduates, and students at a formal poster session the advantages of microbubble technology as an alternative method to traditional chemotherapy, as well as its potential in thrombolysis and gene therapy.

*Note: The bibliography for this project is not attached; it is a separate document also uploaded onto this website.

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  • 1. Christopher PlunkettChristina Amaral[15]
  • 2. AbstractUltrasound, which originated as a medical imaging modality, has sincebeen discovered as a therapeutic tool to treat the diseases that it wasonce only able to observe and diagnose. A recent application ofultrasound lies with microbubble technology. Microbubbles are small(on the order of micrometers), gas-filled spheres that act as contrastagents by scattering an ultrasound signal. Current research involvesthe loading of microbubbles with drugs and injecting them into thebody to be activated by localized ultrasound. Through a process calledcavitation, cell membranes are permeated and the microbubblesrupture, releasing their payload into the intended target tissue.Microbubble destruction via ultrasound has shown success clinically bydelivering chemotherapeutics and anti-cancer drugs to tumor sites inanimals and slowing tumor growth rate, and it holds promise in theareas of gene therapy and thrombolysis. As a relatively newtechnology, ultrasound mediated drug delivery still has limitations thatit must overcome before being used regularly to treat diseases inhumans.[16]
  • 3. French physicistCurie discoverspiezoelectricityFrench physicist Langevinpermitted bygovernment to createdevice to detectsubmerged enemysubmarines; basis forsonar in WWIIAustrian physicianDussik first to use US inmedical diagnosis forbrain tumors; called“hyperphonography”Radiologist Howrycreates B-modeequipment forcross-sectionalanatomical imagesSwedish physicistHertz andcardiologist Edlerbecome “fathers ofechocardiography”“Sonic Boom”2D echo and pulsedDoppler introducedFirst proposed useof microbubbles asa contrast agentfor ultrasound byGramiak and Shah[2]Real-time USdeveloped, smallerprobes andincreased imageresolutionDrug delivery usingmicrobubbles as avector proposed byMiyazaki et al. [3]Unger et al. first toincorporate drugsinside microbubblegas core [3]Note: Uncited events taken from source [1]. [17][18]
  • 4. Ultrasound Design• Ultrasound technology involves the use of 20kHz orgreater frequency pressure waves [3].• In medical applications, waves are produced andmeasured by piezoelectric materials. Thesematerials are crystals and two notable examples arelead zirconate and lithium niobate. These materialsproduce mechanical stress in response to a voltageand vice versa [4].• In ultrasound, piezoelectric materials are idealtransducers. By oscillating the voltage in contactwith the material, a mechanical wave can beproduced. However, the reflected pressure wavescan mechanically stimulate the material causing avoltage to be produced[4].• Typically in medical imaging, a pulse is sent from thepz material (Fig 2.) through a couplant (usually gel)and into the body. Waves that contact a denseobject are reflected back to the transducer. Theresulting voltage is interpreted by a digitalreceiver[4].• In medical imaging, an ultrasound device recordsthe voltage produced by the piezoelectric materialand interpret that into an image. Drug deliveryhowever, is not as focused on what is reflected, butrather what the waves can do inside the body.Figure 1 shows atransducer that wouldbe found in anultrasound device. Itis piezoelectric.Figure 2 shows a typical transducer design.Note the crystal and electrodes at the frontof the model.[20][19]
  • 5. Ultrasound Wave Analysis• Sound waves are variations in pressure overtime. Therefore the plot of an ultrasoundwave has an amplitude of pressure (Pa) anda frequency over time (s) [4].• Wave frequency (f) is equivalent to thenumber of cycles per second of the wave.Wave period (T), or the inverse offrequency, is the number of seconds percycle.• Amplitude of an ultrasound wave in termsof stress (σ) is related to the strength of thepulse. An assumed amplitude is shown inour data table.• In drug delivery, high frequency ultrasoundis necessary. Therefore, this poster willfocus on frequencies of 1 to 10MHz.• A general equation for an oscillating wave isa(t)=σsin(wt) where σ is the amplitude, w isthe product of frequency and 2π, and t isthe elapsed time [4]. A model wave isplotted with an amplitude of 0.1MPa and afrequency of 3.28MHz [5]. This simulates apossible pulse used during microbubbletherapy.Frequency (MHz) 3.28Amplitude (MPa) 0.1W (rad/s) 24001767.87Sampling Frequency (MHz) 76400000Sampling Period (s) 1.3089E-08AssumptionsAssumptions on amplitude and frequencytaken from [5]Time (s) Amplitude (Pa)0 01.3089E-08 30901.699442.6178E-08 58778.525233.9267E-08 80901.699445.2356E-08 95105.651636.5445E-08 1000007.8534E-08 95105.651639.1623E-08 80901.699441.04712E-07 58778.525231.17801E-07 30901.699441.3089E-07 -3.21574E-111.43979E-07 -30901.699441.57068E-07 -58778.525231.70157E-07 -80901.699441.83246E-07 -95105.651631.96335E-07 -1000002.09424E-07 -95105.651632.22513E-07 -80901.699442.35602E-07 -58778.525232.48691E-07 -30901.699442.6178E-07 6.43149E-11Data for One Cycle
  • 6. Microbubble and the Outer Shell• Microbubbles are small gas and lipid spheres that are measured inmicrometers (μm) [6].• There are two components of a microbubble: the outer shell andinner core. The outer shell can be made of rigid or flexiblematerials depending on the design. The core is traditionally aperflurocarbon (PFC) gas. Current FDA approved microbubbleshave a lipid coating [7].• Outer shell design must balance stability with flexibility to achieveoptimal results. Rigid shells will reduce the echogenic effects ofthe microbubble. However, thin shells can allow gas to diffuse outof the bubble. In addition, the shell must be biocompatible [8].• A thin lipid shell (Fig. 1) significantly reduces pressure within thebubble and is biocompatible. However these shells will leak gas.Therefore a water insoluble gas is essential in these types of shells[8]. Figure 2: Optison enhanced image [GE]Figure 1: Electronmicroscope imageof a Lipid coatedMicrobubble.• Albumin shell coatings are more rigid than lipids and canelicit an immune response. Despite this albumin shellshave been used in FDA approved products such asOptisonTM (fig. 2) by GE [6].• A thick shell such as a synthetic polymer will retain thecore and thus a water insoluble gas is not required.However, to act as a contrast agent, the shell must bedestroyed by high frequency ultrasound [6].Figure 3:Comparison ofLipid and Polymermicrobubbles [7][21][22][23]
  • 7. The Core and Laplace Pressure• Initially microbubble cores were filled with air.However, bubble leaks caused excess air in thebloodstream, leading to complications [3].• Currently the inner portion of the microbubblecontains a gaseous PFC core. PFC’s have lowsolubility, which results in longer circulationperiods, and are compatible with the humanbody in small amounts. It is because the PFC ishighly echogenic (can easily return ultrasoundsignals to a source), that it is ideal for use inmicrobubbles as a contrast agent [7].• Research is being conducted in alternatives toPFC cores, including hybrid cores that changephase based on acoustic energy. These phasechange contrast agents are promising but yet tobe FDA approved [7].Phase change agents are prepared with a gaseous PFCcore. Low temperature and high pressure causecondensation of the microbubble. Acoustic energyfrom the ultrasound wave causes vaporization thatincreases volume and echogenicity.[7]
  • 8. Clinical Relevance: Contrast Agents• Ultrasound using microbubble contrast agents is an inexpensive, non-invasive, and fast method foranalyzing blood flow in the body.• A dose of microbubbles ranges from 108 to 109 bubbles. An injection of this dose is placed into aperipheral vein where it quickly spreads throughout the body [9].• The wave scattering ability of a gas is substantially greater (by a factor of 10,000) than that of blood.Therefore microbubbles containing a gas are excellent at scattering an ultrasound signal [9].• Microbubbles are detectable by their own resonance frequency. The application of acousticpressure waves causes the bubble to oscillate at a unique frequency which the ultrasound device candetect. Through signal processing, the ultrasound device can focus on the area surrounding thisfrequency. This is beneficial in capillaries where few microbubbles can be present at one time [9].Contrastenhancedultrasoundimages. Noticethe cleardistinctionbetween tissues.[24] [25]
  • 9. Cavitation and Drug Delivery• Ultrasound alone induces cavitation—the formation,oscillation, and destruction of gas-filled bubbles—due tohigh and low pressures of the wave [2]. The presence ofmicrobubbles can lower the cavitation threshold andincrease cell permeability [2].• Cavitation of microbubbles creates microstreams ofextracellular fluid that exert a shear stress on surroundingcells and perforate them [soft matter]. The microbubblesare also ruptured by the ultrasound wave and release theirpayload into the open target site [6].• Other methods of drug delivery include endocytosis ofmicrobubbles and fusion with the cell membrane [2].Diagram ofthe behaviorofmicrobubbles at differentpoints alongtheultrasoundwave [3].Ultrasound mediated destruction of amicrobubble with externally mountedpayload [6].[3][6]
  • 10. Microbubble Transport through a CapillaryAssumptions takenfrom “Physics ofthe Body” [10].Length of Capillary (m) 1.00E-03Viscosity of Blood (Pa*s) 3.50E-03Velocity of Blood (m/s) 1.00E-03Arterial end blood pressure in a capillary 4.00E+03AssumptionsVolume Flow Rate (m3/s) Change in Pressure(Pa) Venous End Pressure (Pa)7.85E-16 112000 -1.08E+053.14E-15 28000 -2.40E+047.07E-15 12444.44444 -8.44E+031.25664E-14 7000 -3.00E+031.96E-14 4480 -4.80E+022.83E-14 3111.111111 8.89E+023.85E-14 2285.714286 1.71E+035.03E-14 1750 2.25E+036.36E-14 1382.716049 2.62E+037.85E-14 1120 2.88E+039.50E-14 925.6198347 3.07E+031.13E-13 777.7777778 3.22E+031.33E-13 662.7218935 3.34E+031.54E-13 571.4285714 3.43E+03
  • 11. Drug Attachment• Various approaches have been tested for the attachment ofdrugs to microbubbles for targeted delivery in the body. Drugscan be contained within the gas core, which reduces the risk ofpremature release [3]; however, interactions between theperfluorocarbon core and the drug create complications [6].• Incorporation of drugs into microbubblemembranes uses noncovalent bonding and dependsupon charge. This technique is particularly usefulwhen implanting large, negatively charged DNAmolecules into a positively charged membrane [2].• A favorable method is to incorporate drugs into theouter shell of the microbubble for timely releaseupon cavitation by ultrasound. This technique istailored for certain drug properties, such ashydrophilicity and lipophilicity [2]. For instance, oilintroduced into lipid shells was used to dissolve andsuccessfully release chemotherapeutics in vitro [6].[3][26]
  • 12. “Smart” Microbubbles: Target SpecificityThe process of loading microbubbleswith a specific payload, injecting theminto a vein in the general vicinity of atarget site, and locally applyingultrasound leaves room for error. Uponentering the bloodstream,microbubbles are capable of travelingthrough the entire systemic circulationand settling in the incorrect site [2].A solution to the circulation problem iscreating targeted microbubbles that haveligands integrated in their shells. The ligandsare specific and bind to receptors on thesurface of target cells, for instanceendothelial cells or thrombi [2]. They areattached to the microbubble shell bycovalent bonding or interaction of biotin andavidin proteins. Types of ligands includevitamins, antibodies, and peptides [6].Studies show that once themicrobubble has successfullyattached to its target cell, shearstress exerted by blood flow wasnot enough to break theconnection; hence successfulpayload uptake was observed [2].Two types of ligand binding: (a) small hydrophilic ligands and (b) large protein ligands linked by biotin andavidin [6].[6]
  • 13. Clinical Relevance: Cancer Treatment via DrugDelivery• Selective drug delivery is a preferred model forchemotherapy as it specifically targets tumor cellsand reduces unnecessary cell destruction from thedrugs. Microbubbles are ideal vectors as they cancontain a drug payload until treated with ultrasound.• Initial animal tests show significant decrease in tumorsize using ultrasound activated microbubbles (Fig 1).Tumor area decreased to less than half its originalarea after treatment with this method. Traditionalchemotherapy using Genexol PM (GEN) showedtumor reduction at a slower rate than the ultrasounddrug infused microbubble combination (Fig 2) [12].• A control group of empty microbubbles treated withultrasound did not respond to the treatment (Fig 2.)Thus it is the drug microbubbles that cause celldeath.• After the first round of treatment, tumor growthreemerged most likely because of drug resistanttumor cells. Future focus should be placed onrefining current drug options to ensure full tumordestruction [12].Figure 1: Ovarian tumor growth. “US”signifies area of ultrasound treatment[12].Figure 2: A.Ovarian tumorsize (noteempty dropletcontrol) B.Breast cancertumor size[12].[13][13]
  • 14. Thrombolysis, or clot lysis, is the process of breaking down a blood clot bydegrading fibrinogen and fibrin, therefore freeing an occluded vessel [2]. Cavitationof microbubbles due to ultrasound creates a shear stress that disrupts the clot andenhances thrombolysis. Targeted microbubbles bearing thrombolytic agents areespecially powerful in their ability to deliver the clot-dissolving drugs directly to thetarget site [6]. Cavitation induced by low frequency, high power ultrasound alsogreatly improved thrombolysis [2].The use of microbubbles to aidthrombolysis is a promising technique,for it can prevent cardiovasculardiseases caused by blood clots.However, this concept is still in theearly stages of experimentation andhas not yet been applied clinically dueto potential dangers, such as how themicrobubbles will interact with livingcells [2].Microbubblewith ligandsthat bind toreceptors onthrombusand releasethrombolyticagents tobreak up theclot [2].[2][27]
  • 15. A Look to the Future: Gene Therapy• Unlike viral vectors, microbubbles are generallynon-immunogenic. Genes contained in the gascore or outer shell typically remain undigested bythe body and have a better chance of beingdelivered to the target site [2].• Microbubbles show sufficient transfection (geneuptake). Though dependent on the duration andfrequency of the ultrasound wave (Fig 2),expression of a target gene was observed whenusing microbubbles in mice [13].• Studies have incorporated DNA vectors into thealbumin- and lipid-based shells of microbubblesfor gene transfection [6]. DNA can be efficientlybound to microbubbles in concentrations up to100µg DNA per 1E9 microbubbles [13] .Cancer treatment is one application of microbubblegene therapy. Herpes simplex virus-1 thymidinekinase (TK), which can destroy target cells, can bedelivered via microbubbles to a tumor. Current data(Fig. 1) indicates that, with microbubble delivery,there is a significant slowdown of tumor growth [13].Figure 1(right):Triangles representcontrol data whilesquares representmicrobubblestreated with TK [13]Figure 2(left): Table displaying variousgene therapy related studies involvingmicrobubble vectors. All outcomes showsignificant gene uptake and tissueresponse to the gene therapy [2].[13][2]
  • 16. Ethical Issues Concerning Microbubbles• Mechanical stress from microstreams upon cavitation of microbubbles can lead to cellinjury or death. Hemolysis, the destruction of red blood cells, is one example.Cavitation increases the formation of free radicals, highly reactive molecules that cancause cell mutation [2].• The use of microbubbles with ultrasound is tested on non-human subjects before it canbe applied clinically. Experiments are performed on animals such as rats, mice, andrabbits [6]. Whether it is a painful experience for the animal or if painkillers are used isan issue often voiced among animal rights activists, and it raises the question ofwhether animals should suffer for human benefit [14].• Large doses of microbubbles, on the order of 109 microbubbles/mL, were needed forsuccessful drug uptake in animal studies; this amount can be harmful to humans.Ligands attached to the microbubble surface have the potential to bind to areas otherthan the target site, and drug payloads can be washed out by systemic circulationbefore reaching their destination. The small circulation times of microbubbles presentanother challenge. Sonovue, for example, has a half-life of only 6 minutes, and multiplecirculation trials are necessary for successful uptake [6]. [28]