The Use of Ultrasound and Micelles in Cancer Treatment

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331

REVIEW

Copyright copy 2008 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofNanoscience and Nanotechnology

Vol 8 2205ndash2215 2008

The Use of Ultrasound and Micelles in Cancer Treatment

Ghaleb A Husseini12lowast and William G Pitt21Chemical Engineering Department American University of Sharjah Sharjah United Arab Emirates

2Department of Chemical Engineering Brigham Young University Provo Utah 84602 USA

The high toxicity of potent chemotherapeutic drugs like Doxorubicin (Dox) limits the therapeuticwindow in which they can be applied This window can be expanded by controlling the drug deliveryin both space and time such that non-targeted tissues are not adversely affected Recent researchhas shown that ultrasound (US) can be used to control the release of Dox and other hydrophobicdrugs from polymeric micelles in both time and space It has also been shown using an in vivo rattumor model that Dox activity can be enhanced by ultrasound in one region while in an adjacentregion there is little or no effect of the drug In this article we review the in vivo and in vitro researchbeing conducted in the area of using ultrasound to enhance and target micellar drug delivery tocancerous tissues Additionally we summarize our previously published mathematical models thatattempt to represent the release and re-encapsulation phenomena of Dox from Pluronicreg P105micelles upon the application of ultrasound The potential benefits of such controlled chemotherapycompels a thorough investigation of the role of ultrasound (US) and the mechanisms by whichUS accomplishes drug release andor enhances drug potency Therefore we will summarize ourfindings related to the mechanism involved in acoustically activated micellar drug delivery to tumors

Keywords Targeted Delivery Polymeric Micelles Ultrasound Drug Delivery Cancer

CONTENTS

1 Introduction 22052 Ultrasonic Drug Delivery 2205

21 Ultrasound 220522 Mechanisms of Bio-Interactions 220523 Ultrasound-Assisted Drug Delivery 2206

3 Micellar Drug Delivery 220731 Micelles 220732 Liposomes 2207

4 Role of Ultrasound in Producing Drug Release from Micelles 220741 Micellar Drug Carrier 220742 Ultrasonic Drug Release 220843 Stabilized NanoDelivtrade Drug Carrier 2209

5 In Vitro Research and Mechanism of Ultrasound-CellInteraction 2210

6 In Vivo Experiments on Ultrasonically-Assisted Drug Delivery 22127 Conclusion 2213

Acknowledgments 2213References and Notes 2213

1 INTRODUCTION

This review article pertains to the use of ultrasound (US)to release chemotherapeutic drugs from nanometer-sizedcarriers thus targeting the released of drugs to the tissueinsonated by the ultrasound We will start our review with

lowastAuthor to whom correspondence should be addressed

a background on the nature of ultrasound and its use intherapy

2 ULTRASONIC DRUG DELIVERY

21 Ultrasound

Ultrasound consists of pressure waves with frequenciesgreater than 20 kHz usually generated by piezoelectrictransducers that change an applied voltage waveform intomechanical translation of the face of the transducer Likeoptical or audio waves ultrasonic waves can be focusedreflected and refracted and propagated through a mediumthus allowing the waves to be directed to andor focusedon a particular volume of tissue1 The technology for ultra-sonic wave control and delivery are well advanced andpervasive in the areas of biomedical imaging and flowmeasurement Ultrasound is also used in hyperthermic can-cer therapy for breast and other cancers2ndash5 and in physicaltherapy for warming tissues6 The main advantage of ultra-sound is its non-invasive nature the transducer is placed incontact with a water-based gel on the skin and no inser-tion or surgery is required

22 Mechanisms of Bio-Interactions

The interactions of ultrasound with biological tissuesare divided into two broad categories thermal and

J Nanosci Nanotechnol 2008 Vol 8 No 5 1533-4880200882205011 doi101166jnn2008225 2205

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331R

EVIEW

The Use of Ultrasound and Micelles in Cancer Treatment Husseini and Pitt

non-thermal effects Thermal effects are associated withthe absorption of acoustic energy by the fluids or tissuesand are reviewed by others17 Non-thermal bio-effects aregenerally associated with oscillating or cavitating bubblesbut also include non-cavitation effects such as radiationpressure radiation torque and acoustic streaming7 Withrespect to drug delivery these latter effects are proba-bly not involved except to the degree that fluid or parti-cle motion (via acoustic streaming or radiation pressure)increases convection and transport of drug Bio-effectsrelated to cavitation bubbles can produce strong stresseson cells which may increase drug interactions with thecell including increased transport toward and into the cell

Cavitation occurs as gas bubbles oscillate in size inresponse to the surrounding oscillating pressure Ultra-sound excites all sizes of bubbles but those bubbles whosesize imparts a natural resonance frequency near or match-ing the frequency of the acoustic field will achieve thehighest amplitude of oscillation As the acoustic pressureincreases or as the size of the bubble approaches the res-onance size the oscillations become non-linear and even-tually can result in the total collapse of the bubble as theinertia of the inward-moving water surface overcomes theinternal pressure of the bubble This collapse event knownas inertial cavitation causes shock waves creates pres-sures on the order of 100 atm and produces temperatureson the order of several thousand degrees K These violentevents and the radicals generated by the high tempera-tures can damage and even destroy cells7 However eventhe non-inertial (stable) cavitation in which bubbles oscil-late without collapsing can cause significant bio-effectsThe rapidly oscillating surfaces of the bubbles create high

Dr Ghaleb A Husseini has been an Assistant Professor of Chemical Engineering at theAmerican University of Sharjah since January 2004 Dr Husseini graduated with a PhD inChemical Engineering (biomedical engineering emphasis) from Brigham Young UniversityProvo UT in August 2001 His PhD research involved sequestering chemotherapeuticagents (namely Doxorubicin and Ruboxyl) in stabilized Pluronic micelles After graduationhe conducted research in the area of photochemical lithography and soft lithography Hisprojects aimed at creating acid chloride and epoxide functionalized surfaces by exposureto UV light He also developed a novel method of micro-printing on silicon surfaces usingscribed silicon wafers (soft lithography) He has started research in the area of non-viralgene delivery using ultrasonic power Membership in Learned Societies American Instituteof Chemical Engineers Society For Biomaterials Controlled Release Society

Dr William G Pitt received a PhD in Chemical Engineering in 1987 from the Universityof Wisconsin-Madison He obtained a faculty position at Brigham Young University in theChemical Engineering Department where he has served since 1987 He is currently the PopeProfessor of Chemical Engineering at BYU and is an Adjunct Research Professor in theBioengineering Department of the University of Utah During his years at BYU his researchhas spanned many disciplines ranging from biomedical material surfaces and compositematerials to his current work in polymeric-based chemical sensors and ultrasonic enhanceddrug and gene delivery With colleagues and students at BYU and other institutions he hasover 100 peer-reviewed journal publications He holds 6 patents with several others pendingMembership in Learned Societies American Institute of Chemical Engineers Society forBiomaterials Controlled Release Society American Association for Cancer Research

fluid shear stresses that can shear cell membranes makingthem more permeable to small molecules or even disrupt-ing their membranes8ndash10 The existence of cavitation canbe detected by several techniques including the measure-ment of subharmonic and ultraharmonic oscillations11ndash13

trapping of free radicals sonoluminescence and more14

A decrease in acoustic-related effects as ambient pressureincreases is also used to verify the occurrence of cavitationbecause the increased pressure compresses the bubbles andsuppresses the amplitude of oscillation11121415

23 Ultrasound-Assisted Drug Delivery

During the past decade ultrasound has been investigatedby several groups as a potential facilitator of the deliv-ery and uptake of drugs1617 Early studies on transdermaldrug delivery using higher frequencies available in diag-nostic equipment had limited success18ndash21 but by usinglower frequencies (20 kHz) Mitragotri achieved trans-dermal delivery of medium molecular weight proteins(insulin interferon and erythropoeitin)20 His hypothesiswas that cavitation events disrupted the stratum corneumand that such cavitation was more prevalent at lower fre-quencies In our work we believe that cavitation dis-rupts micelles leading to drug release22ndash25 Kruskal et alreported that higher frequency ultrasound (imaging fre-quencies) increased the permeability of blood vessels andincreased the quantity of Dox delivered by stable lipo-somes to hepatic colorectal metastases in a mouse model26

Thus US may increase even further the enhanced perme-ability of tumor capillaries which already allow passive tar-geting of tumors by drugs27ndash29 Kwok et al demonstrated

2206 J Nanosci Nanotechnol 8 2205ndash2215 2008

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331

REVIEW

Husseini and Pitt The Use of Ultrasound and Micelles in Cancer Treatment

ultrasonic-activated release of insulin from a monolithicdrug reservoir with an impermeable surface coating that isdisrupted by the action of ultrasound30 After insonationis stopped the coating reforms and blocks further releaseof drug Ultrasound is credited with causing or enhanc-ing chemical reactions that can be chemotherapeutic31ndash36

Additionally ultrasound has been shown to aid in thedelivery of therapeutic drugs to the brain by causing tran-sient disruptions in the blood brain barrier (BBB)37 Theeffect on the BBB was increased further when microbub-bles were used in conjunction with US

3 MICELLAR DRUG DELIVERY

Several molecular vehicles have been used to deliver thera-peutic drug to the body These include liposomes micellesshelled vesicles solid lipid particles and others38 Thisreview will focus on the use of polymeric micelles andtheir role in acoustically activated drug delivery to cancer-ous tissues

31 Micelles

A micelle consists of an assembly of amphiphilic moleculesarranged to form a hydrophobic core and a hydrophiliccorona The hydrophobichydrophilic interactions of themolecules control the structure of the micelle Hydropho-bic drugs are able to penetrate and accumulate inside thehydrophobic core of these micelles Their sequestrationminimizes the drug interactions with the outer aqueousenvironment Polymeric micelles have a diameter of about20 nm and are considered to have several advantages overother types of drug carriers39ndash53 including(1) Structural stability some polymeric micellesdissociate slowly at levels below their critical micelleconcentration (CMC) requiring hours to days instead ofmilliseconds typical for dissociation of micelles composedof low molecular weight surfactants 404143ndash4850ndash53

(2) Prolonged shelf life(3) Long circulation time in blood and stability in biolog-ical fluids(4) An appropriate size to escape renal excretion(5) An appropriate size to allow extravasation at the tumorsite(6) Simplicity in drug incorporation in comparison withcovalent bonding of the drug to the polymeric carrier(7) Drug delivery independent of drug character54

Polymeric micelles are considered to be much morestructurally stable than micelles formed by low molecu-lar weight compounds Examples of polymeric micellesare those formed by Pluronicreg block copolymers whichare triblock copolymers of poly(ethylene oxide) (PEO) andpoly(propylene oxide) (PPO) often denoted by PEO-PPO-PEO Pluronicreg compounds have gained special attentionin cancer drug delivery because of their ability at low

concentrations to sensitize multi-drug resistant (MDR)cancer cells55 Additionally Pluronicreg compounds havelow in vivo toxicity55

32 Liposomes

There are many publications regarding the delivery of Doxand other drugs from liposomes which are often larger(sim005 to 1 m) than micelles (sim5 to 50 nm) and whichposses a lipid bilayer encapsulating and sequestering watersoluble drugs until release27 Liposomes that are maskedwith PEO remain in circulation longer than those withoutPEO56 There are some reports on the use of ultrasoundto release drugs from liposomes by disrupting and spillingtheir contents57ndash59 A disadvantage of using liposomes isthat they are more difficult to prepare and the drug is notre-encapsulated when the insonation is stopped as it iswith a polymeric micelle system Liposomal drug deliveryis beyond the scope of this review article

4 ROLE OF ULTRASOUND IN PRODUCINGDRUG RELEASE FROM MICELLES

41 Micellar Drug Carrier

As mentioned above micelles are currently being inves-tigated as drug delivery vehicles The most importantmicelles used in drug delivery are made of a hydrophilicPEO and a hydrophobic portion (usually PPO or someother polymer) which allows for their spontaneous assem-ble in water eventually forming a spherical micelle witha hydrophobic core The most common copolymers usedin acoustically activated drug delivery belong to thePluronicreg family of triblock copolymers eg P105 F127P85 L61 etc In P105 the most widely used Pluronicreg forultrasonic drug delivery the number of monomer units ofPEO and PPO are 37 and 56 respectively which creates aweight fraction of approximately 50 PEO and 50 PPOThis surfactant was found to be an ideal drug carrier forultrasonic-activated drug release for several reasons(1) It forms micelles quickly upon simple dissolution inwater(2) The core of PPO is sufficiently hydrophobic to stabi-lize the micelle and sequester hydrophobic drugs60

(3) The micelles can be perturbed by low frequency ultra-sound to release the drug23

(4) The drug is quickly re-encapsulated in the carrierwhen insonation is stopped22

(5) At low concentrations Pluronicreg compounds are non-toxic and can be cleared by the kidneys61

Other Pluronicreg compounds have been investigated asdrug delivery vehicles but were found to be less satis-factory when used as pure Pluronicreg compounds (not inmixed micelles) because those with longer PEO blocks hadtoo high of a critical micelle concentration and those with

J Nanosci Nanotechnol 8 2205ndash2215 2008 2207

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331R

EVIEW

The Use of Ultrasound and Micelles in Cancer Treatment Husseini and Pitt

longer PPO blocks could not dissolve easily in water62

Thus the composition of Pluronicreg P105 appears to beclose to optimal for drug sequestration and ultrasonicrelease

42 Ultrasonic Drug Release

Proper quantification of the amount of drug release frommicelles is essential in these studies To this end we devel-oped a laser fluorescence detection system to quantifythe amount and the kinetics of Dox release from thesemicelles22ndash2563 The system consists of an argon-ion laserat 488 nm directed into a glass cuvette containing the trialsolution to be insonated A fiber optic probe is used tocollect the fluorescence emission from the cuvette Thecollected light passes through a bandpass filter centeredat 535 nm to a sensitive light detector whose signal isdigitized and stored on a computer The temperature ofthe ultrasonic exposure chamber is maintained at 37 Cby a recirculating thermostatic bath A decrease in flu-orescence is attributed to Dox being released from themicelle core to the aqueous phase and the release wasquantified using a calibration with free Dox2324 in whichDox dissolved in PBS simulated 100 release Becausethe emission from Dox is quenched by water the mea-sured fluorescence decreased as Dox is transferred fromthe hydrophobic core of the micelle to the aqueous phaseResults using this system revealed that up to 10 of theDox is released depending upon the insonation intensityand frequency22ndash2563 Drug release was also observed at20 kHz (005 Wcm2) Pulsed insonation resulted in pulseddrug release and re-encapsulation23

The release of Dox from Pluronicreg micelles was stud-ied as a function of ultrasonic frequency and intensityThere are several aspects of Dox release at 20 kHz and70 kHz that merit discussion First the same level of drugrelease could be attained at both frequencies but muchless acoustic intensity was required at the lower frequencyto produce the same amount of release This is consistentwith the hypothesized cavitation mechanism since bubbleamplitude and cavitation activity in general increases asfrequency decreases64 The alternative hypothesis that theobservation could also be attributed to a greater populationof bubbles of near-resonant size at the lower frequencywas discounted because the resonant sizes of air bubblesat 20 and 70 kHz are 0165 and 0047 mm respectivelyand bubbles of the larger size were never observed in theexperiments

In an attempt to study the mechanism of ultrasonicdrug release from Pluronicreg micelles we improved thedetection technique by installing a coaxial cable that wascapable of both directing the laser light into the sam-ple and collecting the emissions24 The acoustic spec-tra were collected at the different power densities Theresults of these experiments showed the existence of a drug

Fig 1 An illustration of the proposed mechanism of ultrasonic releaseof Dox from Pluronic micelles

release threshold (ranging between 035 and 040 Wcm2at 70 kHz These intensities are near the threshold for theonset of collapse cavitation indicating that this type ofevent is a required element for drug release from thesemicelles Furthermore the onset of drug release corre-sponded to the emergence of subharmonic peak in theacoustic spectra which is indicative of the onset of collapsecavitation We hypothesized that shock waves caused bythe collapse of cavitating bubbles are capable of perturbingthe micelle structure enough for the drug to be released asillustrated in Figure 1 Equally interesting was the obser-vation of similar release thresholds with two other micellarsystems namely NanoDelivtrade (P105 micelles stabilized byan interpenetrating network see Section 43) and PNHL(a copolymer consisting of a PEO block covalently bondedto a copolymer containing poly(N-isopropyl acrylamide)and polylactate esters of hydroxyl-ethyl methacrylate)65

While the observed amount of release was less than fornon-stabilized micelles the first measurable release wasalso correlated with the appearance of a subharmonic peak

Recently Stevenson-AbuoelNasr et al developed a newkinetic model to account for the triphasic nature of therelease profile66 The new model introduced 5 differentconstants in an attempt to capture the complex behaviorof release namely the rate of micelle destruction micelleassembly drug re-encapsulation nuclei destruction and themaximum amount of Dox that micelles can hold Addi-tionally the model used a size distribution of micellesand it predicted the larger micelles to be destroyed firstwhich would in turn cause a fast release phase This phaseis followed by a slower phase where smaller micelles aresheared and broken up Finally a third phase was mod-eled in which the smaller fragments and smaller micellescoalesce into larger micelles The advantage of this modelis that it incorporates the various phases of the cavita-tion phenomena into kinetic calculations thus introducing

2208 J Nanosci Nanotechnol 8 2205ndash2215 2008

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331

REVIEW

Husseini and Pitt The Use of Ultrasound and Micelles in Cancer Treatment

a more accurate representation of the physical mechanisminvolved in drug release

Artificial neural networks (ANN) were also used to cap-ture the non-linear nature of release67 Previously collectedrelease data were compiled and used to train validateand test an ANN model Sensitivity analysis was then per-formed on the following operating conditions ultrasonicfrequency power density Pluronicreg P105 concentrationand temperature The model predicted that drug releasewas most efficient at lower frequencies As expected therelease increased as the power density increased Sensi-tivity plots of ultrasound intensity reveal a drug releasethreshold of 0015 Wcm2 and 04 Wcm2 at 20 and70 kHz respectively Based on the developed model Doxrelease is not a strong function of temperature suggestingthat thermal effects do not play a major role in the physi-cal mechanism involved Finally sensitivity plots of P105concentration indicates that higher release was observed atlower copolymer concentrations The ANN model is cur-rently being used to develop a controller that can optimizethese ultrasonic parameters in a clinical setting

43 Stabilized NanoDelivtrade Drug Carrier

The difficulty with using Pluronicreg micelles in vivo isthat the micelles are diluted below their CMC wheninjected into the blood stream thus dissolving and pre-maturely dropping their load of drug Therefore micellesused in tumor targeting were stabilized by polymerizingan interpenetrating network (IPN) of thermally sensitiveacrylamide in the hydrophobic core In this synthesis a10 wt solution of P105 was placed in a round-bottomedflask and 05 wt NN-diethyl acrylamide (NNDEA) wasadded along with azobisisobutyronitrile (AIBN) as an ini-tiator (0001 wt) and bisacryloyl cystamine (BAC) as thecrosslinker (01 wt) The solution was heated to 65 Cunder nitrogen for 24 hrs resulting in the polymerizationof a crosslinked interpenetrating network entangling thePPO core of the micelle that prevents the micelle fromdissolving immediately upon dilution62

This stabilized micelle system called NanoDelivtrade hasmany advantages as an ultrasonic-activated drug deliveryvehicle First the NanoDelivtrade still sequesters Dox andreleases it upon insonation22 The only difference betweenPluronicreg and NanoDelivtrade ultrasonic release noted to dateis that at similar intensities NanoDelivtrade released less drugthan did Pluronicreg P105 possibly due to the strongerforces of the covalent network holding the hydrophobiccore together and resisting shear from the shock waves25

Secondly because the NNDEA interpenetrating networkitself is a thermally reversible hydrogel the NanoDelivtrade

core expands below 31 C (thus enabling drug loading atroom temperature) and collapses into a tight hydropho-bic core above 31 C (thus retaining the drug in thehydrophobic core) Dynamic light scattering shows the size

Fig 2 Transmission electron micrograph of NanoDelivtrade particles adja-cent to a DHDK12TRb cell The NanoDelivtrade particles were stainedwith Pb-salicylate before being incubated with the cells grown on a beamcapsule The micrograph was provided by Dr Virginia Bayer of UtahValley University

of a NanoDelivtrade micelle to be about 60 nm at 37 Csmall enough to pass easily through capillaries and beextravasated in leaky tumor capillary networks and yetlarge enough to avoid renal clearance Figure 2 showssome Nanodelivtrade micelles (stained with Pb-salicylate)adjacent to rat colon cancer cells (DHDK12TRb)

NanoDelivtrade micelles are completely degradableBecause the non-toxic Pluronicreg chains are not crosslinkedto the interpenetrating network they slowly diffuse awayand are cleared by the kidneys The remaining IPN willdegrade slowly by the reduction of the disulfide bondsthus releasing non-toxic polyNNDEA chains of about22000 Daltons These chains are short enough to becleared by the kidneys68

Dissolution of NanoDelivtrade is required to prevent itsaccumulation in the body However the NanoDelivtrade mustremain stable long enough to deliver their drug to thetarget tissue and to avoid rapid release of drug into thesystem producing unwanted high concentrations and sideeffects in other tissues For this reason NanoDelivtrade sta-bility was studied with a luminescence spectrometer usingdiphenyl hexatriene (DPH) as a fluorescent probe62

The emission spectrum of DPH is highly dependentupon the hydrophobicity of the local environment and

J Nanosci Nanotechnol 8 2205ndash2215 2008 2209

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331R

EVIEW

The Use of Ultrasound and Micelles in Cancer Treatment Husseini and Pitt

DPH has almost no fluorescence in aqueous solutionswhile it is very fluorescent in hydrophobic environments69

This makes DPH very useful for determining if ahydrophobic environment is present such as in the coreof a micelle A stock solution of DPH in tetrahydrofuran(THF) was added to one mL of the undiluted (10 wtNanoDelivtrade polymerization sample) The sample was thendiluted to 001 wt leaving 01 gml DPH and 005 lTHFml The samples were excited at 360 nm while theemission was measured at 430 nm Preliminary experi-ments demonstrated that the low concentration of THFpresent did not affect the emission intensity

The 001 wt P105 sample showed very low emissionintensity while the 001 wt NanoDelivtrade sample showedhigher emission intensity The DPH emission intensitywas monitored over time and a decline in the emis-sion intensity from the NanoDelivtrade sample was observedover hours The emission intensity appeared to declineexponentially with a half-life of about 17 hours It washypothesized that the decline in emission intensity was dueto disentanglement of Pluronicreg P105 molecules from theinterpenetrating network of poly(NNDEA) Residual emis-sion was attributed to the residual polyNNDEA networkthat did not dissolve in water It did however dissolveupon addition of -mercapto ethanol68

By changing the crosslink density of the interpenetrat-ing network the hypothesis that NanoDelivtrade stability andits rate of dissolution are controlled by the rate of diffu-sion of Pluronicreg chains from the core was tested A seriesof NanoDelivtrade were synthesized with increasing amountsof the BAC crosslinker In theory as crosslink densityincreases the size of the closed rings entrapping Pluronicreg

chains will decrease thus reducing the rate of diffusionout of the stabilized NanoDelivtrade structure NanoDelivtrade

were made with BAC ranging from 0001 to 0167 wtin the polymerization mixture with 05 wt NNDEA68

As the amount of crosslinker increased micellar stabil-ity (as measured by DPH emission) also increased How-ever at the highest concentration the resulting solutionwas cloudy indicating that some crosslinking was occur-ring in the aqueous phase not solely within the micellecore68 The NanoDelivtrade synthesized using 01 wt BACwas used in the subsequent in vivo rat experiments

Recently Rapoport et al made degradable nanoemul-sions of perfluoropentane (PFC5 stabilized withpoly(ethylene oxide) block-poly lactide (PEG-PLLA)70ndash72

At a PFC5 volume concentration of 01 micelles stillexisted in solution with an average size of 215 nm whileat higher PFC5 concentrations (1 vol) no micelles weredetectable since all the polymeric chains were expendedin stabilizing the PFC5 droplet-emulsion At the higherPFC5 concentration two sizes of droplets were observedone at 256 nm and the other at 811 nm The authorsconcluded that the 256 nm particles will be able to pene-trate tumor capillaries while the larger particles too large

to accumulate in cancerous tissues can aid in ultrasonicdrug delivery from smaller droplets by increasing thetumor intracellular uptake upon their transformation tocollapsing gas bubbles during insonation

5 IN VITRO RESEARCH AND MECHANISMOF ULTRASOUND-CELL INTERACTION

Once ultrasonic-activated drug release from micelleswas confirmed we tested its effectiveness on anin vitro cell system HL-60 cells were exposed to Doxand Dox-encapsulated in either Pluronicreg micelles orNanoDelivtrade73ndash75 Cells exposed to encapsulated Dox (bothPluronicreg and NanoDelivtrade) had much longer cell survivalthan cells exposed to free Dox When 70-kHz ultra-sound was applied to the cells those exposed to encap-sulated Dox died much more rapidly than cells exposedto free Dox These results indicated that both Pluronicreg

and NanoDelivtrade sequestered Dox from the HL-60 cellsand increased cell survival compared to survival withfree Dox However insonation appeared to release theDox and quickly kill the cells The protective effect ofNanoDelivtrade lasted about 20 hours similar to the half-lifeof the NanoDelivtrade

Our research group has also measured the uptake of Doxby cancer cells in vitro5476ndash78 Uptake was measured bothdirectly and indirectly In the latter method Dox absorp-tion by cells was calculated by measuring drug depletionfrom the incubating medium (using a spectrofluorometer)In the former method fluorescence of Dox was measuredin cell lysates The cells were lysed in 2 sodium dode-cyl sulfate (SDS) at 37 C for 2ndash3 days with periodicalstirring which results in Dox transfer from the cells tothe SDS micelles Calibration experiments showed lineardependence of Dox fluorescence on Dox concentration inPBS Pluronicreg or SDS micelles The cell concentration inthe lysates was measured by BCA assay76 or by countingcells before lysis using a hemacytometer These experi-ments have quantified Dox uptake kinetics and isothermsin several studies547677 Ultrasound and the presence ofPluronicreg has a large influence on the uptake of Doxby drug sensitive and drug resistant breast cancer cells(A2780 and A2780ADR)79 Insonation increased uptakein both cell lines however there was less uptake from a10 wt Pluronicreg solution than from a 01 Pluronicreg

solution80

The uptake of Dox by HL-60 cells was studied bypulsing 70 kHz US in tone bursts ranging from 01 secto 20 sec in duration while the time between tonebursts varied from 01 to 2 sec in duration The resultsshowed that with constant ldquointer-burstrdquo time and with con-stant total insonation time the amount of uptake by thecells increased with insonation length up to about 3 sec-onds which was the same uptake observed under CWinsonation However the amount of uptake did not depend

2210 J Nanosci Nanotechnol 8 2205ndash2215 2008

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331

REVIEW

Husseini and Pitt The Use of Ultrasound and Micelles in Cancer Treatment

upon the length of the ldquointer-burstrdquo period76 From theseexperiments the time to achieve 90 of full uptake wascalculated to be about 25 sec of insonation

Therefore uptake into the cell proceeds at a slightlyslower rate than release but both are the same order ofmagnitude The important observation is that the amountof cellular uptake was independent of the length of inter-burst time This indicated that the cancer cells did notallow any Dox to go back to the micelles (beyond whatmight have diffused back faster than the shortest inter-bursttime of 01 sec) Although Dox returns to the micelles inthe absence of cancer cells it apparently does not happento a measurable extent in the presence of cells which indi-cates that the cells are very effective at competing with themicelles for the Dox

The fluorescence of Dox makes it ideal for uptake stud-ies using flow cytometry and fluorescence microscopyFor fluorescence microscopy and confocal microscopycells were first fixed with 3 formalin then washed withPBS containing 3 formalin and then sealed on glassslides Confocal microscopy showed different distributionsof Dox within the cell with Dox mostly in the nucleus79

Pluronicreg P105 was then labeled with 56-carboxy-2prime7prime-dichlorofluorescein a pH-sensitive fluor which fluorescesbrighter at pH 74 than in acidic conditions80 HL-60 cellsexposed to these labeled Pluronicreg micelles with or with-out insonation took up the label and were subsequentlystudied with confocal microscopy and flow cytometry Flu-orescence micrographs and confocal microscopy showeddistribution of Pluronicreg to membrane vesicles and thecytosol

Our group examined the hypothesis that US increasesthe rate of endocytosis of micelles into HL-60 cells81 Inthese experiments we used a label (Lysosensor Green) thatfluoresces more strongly in a lysosome or an endosomewhere the pH is more acidic (about 48) compared to apH of 71 outside these compartments8283 Flow cytome-try was then used to analyze cells exposed to US and P105micelles tagged with Lysosensor Green Results showed nosignificant difference in fluorescence between cells incu-bated and ultrasonicated for 1 hour at 70 kHz This mech-anistic study concluded that since ultrasound did not causethe fluorescent probe to partition to a more acidic envi-ronment more than it did without ultrasound (thus enhanc-ing fluorescence) the upregulation of endocytosis is notlikely to be one of the mechanisms involved in US-assistedmicellar drug delivery

While the study above did not show any evidence ofreceptor mediated endocytosis or pinocytosis Rapoportet al84 reported that acoustic stimulus enhanced the rate ofendocytosis of micelles into two human cell lines Sheikovet al later showed that US was capable of promotingpinocytosis in the endothelial cells lining brain arteriolesand capillaries85 Thus the mechanism of this acousticenhancement of drug delivery from micelles is still under

debate and more research was undertaken by our researchgroup to answer this mechanistic question These studiesare summarized below

Stringham et al86 reported that collapse cavitation wasimplicated in colon cancer cell membrane disruptionTheir study showed that calcein uptake was reduced byincreasing the hydrostatic pressure (up to 3 atm) at con-stant ultrasonic intensity and frequency Increasing hydro-static pressure reduced acoustic bubble activity associatedwith collapse cavitation8788 although stable cavitation stilloccurred In these experiments lower membrane perme-ability correlated directly with higher hydrostatic pressureand cavitation suppression supporting the hypothesis thatcollapse cavitation is involved in drug uptake in vitro

We further investigated the mode of death associatedwith cells exposed to a combination of Pluronic micellesUS and Dox2473 Using the comet assay our group stud-ied the electrophoretic pattern of the nuclear DNA fromHL-60 cells sonicated in the presence of 10 P105 anda Dox concentration of 10 gml for 2 hours at 70 kHzThe gradual damage observed after two hours of acousticexposure were consistent with apoptosis as a mode of celldeath rather than necrosis The fact that apoptosis is theprimary mechanism of cell death in acoustically triggeredmicellar drug delivery is important evidence that at theselevels of insonation the Dox released by US is causinggradual DNA disintegration as opposed to the severe cellmembrane damage and necrosis caused by the US damag-ing the cell membrane beyond repair

Howard et al89 studied the effect of ultrasound onpaclitaxel in micelles of methyl-capped poly(ethyleneoxide)-co-poly-(L-lactide)-tocopherol on a breast cancerdrug-resistant cell line Their study used 1 MHz Ultra-sound at a power density of 17 Wcm2 and duty cycleof 33 Without ultrasonication less encapsulated Pacli-taxel accumulated inside the cells when compared to Pacli-taxel administered from a clinical formulation (free ornon-encapsulated Paclitaxel) confirming previous reportsof cell protection from the action of chemotherapeuticagents by encapsulation using polymeric micelles How-ever upon the application of ultrasound drug accumula-tion from encapsulated Paclitaxel drastically increased andsurpassed the amount of drug found in non-insonated cellsThe same study also showed that using the above men-tioned polymeric micelles in conjunction with ultrasoundwas effective in complete tumor regression in nunu miceinoculated with the same drug resistant cell line

All of the above in vitro studies convincingly indicatethat ultrasonic cavitation is producing stress on the cellmembrane in a manner to allow greater drug uptake thanwould occur without ultrasound This data coupled withother studies showing how US creates repairable holes incell membranes90 leads to the conclusion that in vitro USboth releases drug from some types of carriers and createstransient holes in cell membranes through which free or

J Nanosci Nanotechnol 8 2205ndash2215 2008 2211

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331R

EVIEW

The Use of Ultrasound and Micelles in Cancer Treatment Husseini and Pitt

released drug or even micelles can enter into the cellcytosol thus bypassing the endocytotic pathway

6 IN VIVO EXPERIMENTS ONULTRASONICALLY-ASSISTEDDRUG DELIVERY

Once the mechanism of ultrasonically activated drugrelease to cells in vitro was understood it was time toexamine the drug delivery system in vivo A convenientmodel is that of tumor bearing rats and mice The Pittgroup uses a rat model91ndash93 while a mouse model is usedby the Rapoport group729495 and the Myhr group96

In the rat model a colon carcinogen cell line DHDK12TRb was grown in RPMI containing 2 mM nys-tatin 02 mM Gentamicin 2 mM L-glutamine and 20fetal bovine serum Forty-six 6-wk-old BDIX rats wereanesthetized with a combination of ketamine HCl andmedetomidine HCl inoculated in each upper hind legwith a subcutaneous injection of the tumor cell suspen-sion (2times 106 cellsmL) and allowed to recover92 Tumorvolume was estimated by making two perpendicular mea-surements (a and b where a gt b) with a caliper and byusing the formula T V = ab22 The rats were randomlydivided into ten groups including a control group Selec-tion of right or left tumor for US treatment was random-ized Five weeks following tumor inoculation rats werepreanesthetized with an ip injection of ketamine and pre-treated with sq injections of dexamethasone and diphen-hydramine to reduce incidence of anaphylactic shock97

Intravenous administration of free Dox or Dox encapsu-lated in NanoDelivtrade occurred via the tail vein Imme-diately following injection the anesthetic regimen wascompleted and the rats were placed in a special restraintdevice to exposed one depilated leg and its tumor to unfo-cused 20 kHz or 70 kHz ultrasound The rest of the bodyincluding the other leg was not exposed to US The vari-ables investigated in these experiment included appliedpower density (1 and 2 Wcm2 ultrasonic frequency (20and 70 kHz) Dox concentration (133 267 and 8 mgkg)power train (continuous and pulsed) and treatment regi-men (once and twice weekly) US was applied for 1 hrto one leg of the animal and treatment was repeated onceweekly for 4 weeks on the same leg

Dox concentration of 8 mgkg was lethal within twoweeks and doses of 533 and 40 mgkg produced deathwithin 6 weeks93 Lower concentrations of 133 and267 mgkg were not fatal The growth of the bilateraltumors in the negative control groups (no US on anytumor) was relatively similar increasing approximatelyexponentially over time

The tumors that were exposed to ultrasound and encap-sulated Dox however did not generally grow as much asthe non-insonated tumors In fact the US-treated tumorgenerally slowed in growth and sometimes even regressed

A paired comparison of insonated versus non-insonatedtumor size in rats receiving encapsulated Dox (any concen-tration) showed that insonated tumors were significantlysmaller than untreated tumors (p = 00062) A similarpaired analysis of all rats that received encapsulated Doxat 267 mgkg revealed that insonated tumors were againsignificantly smaller than when not insonated (p= 0017)All of the tumors exposed to 70 kHz US and encapsulatedDox during their treatment were significantly smaller thanthe non-treated tumors (p = 0029) The positive controlgroup receiving free Dox showed no statistical differencebut then neither did any other individual treatment groupbecause of the scatter in tumor growth patterns and thesmall sample size

As a continuation of the above work Staples et alinvestigated the exposure of the same tumor cell lineto NanoDelivtrade encapsulated Dox at two US frequen-cies namely 20 kHz and 476 kHz for 15 minutes91 Theinsonated tumors grew more slowly than the non-sonicatedcontrols (p = 00047) However there was no significantdifference in growth suppression when comparing the twodifferent frequencies (p = 093)

Staples also compared Dox bio-distribution in severalorgans at different times after the rats were treated witha combination of DoxNanoDelivtradeUS91 In spite of theslower growth of tumors receiving USDoxNanoDelivtrade

compared to DoxNanoDelivtrade without US their studyfound that shortly after injection and ultrasonic exposure(30 minutes) the insonated tumors contained slightly moreDox than the contra lateral control tumors At longer timepoints (3 hrs and greater) there was a decrease in con-centration with time but there was no difference in Doxconcentration between the insonated and control tumorsDox concentrations deceased with time in all other tissuesinvestigated (the heart kidneys liver and muscle) andwere zero within a week of drug administration

Using a mouse model Gao et al98 studied the intra-cellular distribution of two fluorescently labeled carriersThey insonated (1 MHz) ovarian cancer tumors inoc-ulated in nundashnu mice in the presence of unstabilizedPluronic P105 and Pluronic P105 stabilized using PEG-diacylphospholipid The study showed that insonation for30 s or more was capable of increasing the concentra-tion of these two labeled micelle formulations at the tumorsite Rapoport et al99 showed that the accumulation ofthe micelle was significantly higher in the sonicated tumorthan in the non-sonicated mouse model mentioned aboveTheir study also reported that encapsulated Dox did notaccumulate in the heart which would alleviate the car-diotoxicity of Dox

Myhr et al96 used NanoDelivtrade to encapsulate achemotherapy drug (5-fluorouracil) The authors thenapplied ultrasound to mice inoculated with a humancolon cancer cell line Ultrasound significantly reduced thetumor volume compared to the control group The authors

2212 J Nanosci Nanotechnol 8 2205ndash2215 2008

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331

REVIEW

Husseini and Pitt The Use of Ultrasound and Micelles in Cancer Treatment

also concluded that more significant tumor reductionswere observed when higher drug concentrations wereadministered

7 CONCLUSION

Ultrasound has great potential as a mediator of chemother-apy because it can be non-invasively focused on a tissuevolume within the body and thus can mediate or acti-vate drug delivery to that site only Thus the drug deliverycan be controlled in spatial position and in the timing ofthe delivery Polymeric micelles are ideal for drug deliv-ery because of their size and ease of preparation In thisreview we reported on the application of ultrasound toenhance chemotherapy using a micelle-sized carrier thatcan sequester the chemotherapeutic agent and prevent itsinteraction with the rest of the body but then can releasethe agent at the desired place and time upon the applica-tion of ultrasound

Numerous mechanistic studies have shown that ultra-sonic cavitation is responsible for US-activated drugdelivery In vitro cavitation releases drug from sometypes of micelles particularly those that are Pluronicreg-based Just as importantly cells in vitro are renderedmore permeable to the drugs by cavitation events Itis most likely that shear stresses from collapse cavita-tion events are rupturing micelles and permeabilizing cellmembranes In vivo ultrasonic-activated chemotherapeu-tic delivery from micelles reduces tumors in rats andmice At short times post-insonation there is more drugpresent in the sonicated tumors most probably the resultof cavitation-induced shear of micelles It is highly likelythat the tumor cell membrane is also permeabilized result-ing in greater drug uptake

The advantages of such a delivery system are numer-ous Since the potent drug is sequestered until the desiredrelease place and time the side effects of chemotherapycan be minimized The drug loading in the body could beincreased without detriment and the drug loading will beconcentrated at the targeted site to produce the maximumeffect This technology could also be combined with othernovel targeting techniques such as attachment of tumor-targeting molecules to the outside of the micelles Sucha technology would provide the oncologist with a veryeffective weapon in the fight against cancer

NOMENCLATURE

AIBN AzobisisobutyronitrileANN Artificial Neural NetworksBAC Bis-Acryloyl CystamineBBB Blood Brain BarrierBCA Bicinchoninic AcidCMC Critical Micellar ConcentrationDox Doxorubicin

DPH Diphenyl HexatrieneEPR Electron Paramagnetic ResonanceIPN Interpenetrating NetworkMDR Multidrug ResistanceNIPAAm N-IsopropylacrylamideNNDEA NNprime-DiethylacrylamidePEO Poly(Ethylene Oxide)PEG-PLLA Poly(Ethylene Oxide) block-Poly(L-Lactide)PEG-PLC Poly(Ethylene Oxide) Block-Poly LactidePFC5 PerfluoropentanePluronicreg A Triblock Copolymer of PEO-PPO-PEONanoDelivtrade A Pluronicreg P105 micelle

stabilized with an IPN of NNDEAP105 Pluronicreg P105 (PEO37-PPO56-PEO37PPO Poly(Propylene Oxide)SDS Sodium Dodecyl SulfateUS Ultrasound

Acknowledgments The authors gratefully acknowl-edge financial support from NIH grant CA 98138

References and Notes

1 T G Leighton Prog Biophys Mol Biol 93 3 (2007)2 P E Huber and J Debus Radiat Res 156 301 (2001)3 P E Huber J W Jenne R Rastert I Simiantonakis H-P Sinn

H-J Strittmatter D von Fournier M F Wannenmacher andJ Debus Cancer Res 61 8441 (2001)

4 X Q Lu E C Burdette B A Bornstein J L Hansen and G KSvensson Int J Hyperthermia 12 375 (1996)

5 H R Underwood E C Burdette K B Ocheltree and R L MaginInt J Hyperthermia 3 257 (1987)

6 D O Draper J C Castel and D Castel J Orthop Sports PhysTher 22 142 (1995)

7 W L Nyborg Ultrasound Med Biol 27 301 (2001)8 A R Williams and D L Miller Ultrasound Med Biol 6 251

(1980)9 J A Rooney Ultrasound Its Chemical Physical and Biological

Effects edited by K S Suslick VCH New York (1988) p 6510 J A Rooney Science 169 869 (1970)11 K I Morton G R Ter Haar I J Stratford and C R Hill Br J

Cancer 45 147 (1982)12 K I Morton G R t Haar I J Stratford and C R Hill Ultrasound

Med Biol 9 629 (1983)13 E C Everbach I R S Makin M Azadniv and R S Meltzer

Ultrasound Med Biol 23 619 (1997)14 A A Atchley and L A Crum Ultrasound Its Chemical Physical

and Biological Effects edited by K S Suslick VCH PublishersNew York (1988) p 1

15 M Delius Ultrasound Med Biol 23 611 (1997)16 W G Pitt Am J Drug Deliv 1 27 (2003)17 W G Pitt G A Husseini and B J Staples Expert Opin Drug

Delivery 1 37 (2004)18 J Saxena N Sharma M C Makiod and U V Banakar J Biomat

Appl 7 227 (1993)19 S Mitragotri D Blankschtein and R Langer Science 269 850

(1995)20 S Mitragotri D A Edwards D Blankschtein and R Langer

J Pharmaceuti Sci 84 697 (1995)21 D G Kassan A M Lynch and M J Stiller J Amer Acad

Dermatology 34 657 (1996)

J Nanosci Nanotechnol 8 2205ndash2215 2008 2213

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331R

EVIEW

The Use of Ultrasound and Micelles in Cancer Treatment Husseini and Pitt

22 G A Husseini D A Christensen N Y Rapoport and W G PittJ Controlled Release 83 302 (2002)

23 G A Husseini G D Myrup W G Pitt D A Christensen andN Y Rapoport J Controlled Release 69 43 (2000)

24 G A Husseini M A Diaz E S Richardson D A Christensenand W G Pitt J Controlled Release 107 253 (2005)

25 G A Husseini M A Diaz Y Zeng D A Christensen and W GPitt J Nanosci Nanotechnol 7 1 (2007)

26 J Kruskal S Goldberg and R Kane Novel in vivo use of con-ventional ultrasound to guide and enhance molecular delivery anduptake into solid tumors Annual Meeting of the Radiological Societyof North America RSNA Chicago IL (2001) p 804

27 H Maeda Adv Enzyme Regul 41 189 (2001)28 K Greish T Sawa J Fang T Akaike and H Maeda J Controlled

Release 97 219 (2004)29 A Gabizon R Catane B Uziely B Kaufman T Safra R Cohen

F Martin A Huang and Y Barenholz Cancer Res 54 987 (1994)30 C S Kwok P D Mourad L A Crum and B D Ratner J Biomed

Mater Res 57 151 (2001)31 E Takada M Sunagawa E Ohdaira and M Ido Ultrasound Med

Biol 23 S132 (1997)32 D B Tata G Hahn and F Dunn Ultrasonics 31 447 (1993)33 D B Tata J Biglow J R Wu T R Tritton and F Dunn Ultra-

sonics Sonochem 3 39 (1996)34 A H Saad and G M Hahn Cancer Res 49 5931 (1989)35 A H Saad and G M Hahn Ultrasound Med Biol 18 715 (1992)36 A H Saad and G M Hahn Heat Transfer in Bioengineering and

Medicine edited by J C Chato T E Diller K R Diller and R BRoemer Am Soc Mech Engr Press New York (1987) p 28

37 M Stephen and A Alonso Prog Biophys Molec Biol 93 354(2006)

38 V P Torchilin Adv Drug Delivery Rev 58 1532 (2006)39 J E Chung M Yokoyama M Yamato T Aohagi Y Sakurai and

T Okano J Controlled Release 62 115 (1999)40 K Kataoka G S Kwon M Yokoyama T Okano and Y Sakurai

J Controlled Release 24 119 (1993)41 K Kataoka T Matumoto M Yokoyama T Okano Y Sakurai

S Fukushima K Okamoto and G S Kwon J Controlled Release64 143 (2000)

42 G Kwon M Naito M Yokoyama T Okano Y Sakurai andK Kataoka J Controlled Release 48 195 (1997)

43 G Kwon M Naito M Yokoyama Y Sakurai T Okano andK Kataoka Langmuir 9 945 (1993)

44 G S Kwon M Naito K Kataoka M Yokoyama Y Sakurai andT Okano Colloids Surf B Biointerfaces 2 429 (1994)

45 G S Kwon M Naito M Yokoyama T Okano Y Sakurai andK Kataoka Pharm Res 12 192 (1995)

46 G S Kwon S Suwa M Yokoyama T Okano Y Sakurai andK Kataoka J Controlled Release 29 17 (1994)

47 G S Kwon S Suwa M Yokoyama T Okano Y Sakurai andK Kataoka Pharm Res 12 192 (1995)

48 G S Kwon M Yokoyama T Okano Y Sakurai and K KataokaPharm Res 10 970 (1993)

49 M Yokoyama Advances in Polymeric Systems for Drug Deliveryedited by T Okano Gordon and Breach Science Publishers IverdonSwitzerland (1994) p 24

50 M Yokoyama G S Kwon M Naito T Okano Y Sakurai T Setoand K Kataoka Bioconj Chem 3 295 (1992)

51 M Yokoyama T Okano Y Sakurai H Ekimoto C Shibazaki andK Kataoka Cancer Res 51 3229 (1991)

52 M Yokoyama T Okano Y Sakurai S Fukushima K Okamotoand K Kataoka J Drug Targetting 7 171 (1999)

53 M Yokoyama T Sugiyama T Okano Y Sakurai M Naito andK Kataoka Pharm Res 10 895 (1993)

54 N Y Rapoport J N Herron W G Pitt and L Pitina J ControlledRelease 58 153 (1999)

55 E Batrakova S Lee S Li A Venne V Alakhov and A KabanovPharm Res 16 1373 (1999)

56 M C Woodle Adv Drug Deliv Rev 32 139 (1998)57 S Ning K Macleod R Abra A H Huang and G M Hahn Int

J Radiation Oncology Phys 29 827 (1994)58 S P Vyas R Singh and R K Asati J Microencapsul 12 149

(1995)59 E C Unger T P McCreery R H Sweitzer V E Caldwell and

Y Wu Invest Radiol 33 886 (1998)60 N Munshi N Rapoport and W G Pitt Cancer Lett 117 1 (1997)61 A Venne S Li R Mandeville A Kabanov and V Alakhov Cancer

Res 56 3626 (1996)62 J D Pruitt G Husseini N Rapoport and W G Pitt Macro-

molecules 33 9306 (2000)63 G A Husseini N Y Rapoport D A Christensen J D Pruitt and

W G Pitt Coll Surf B Biointerfaces 24 253 (2002)64 C E Brennen Cavitation and Bubble Dynamics Oxford University

Press New York (1995)65 Y Zeng and W G Pitt J Biomat Sci Polym Ed 16 371 (2005)66 D Stevenson-Abouelnasr G A Husseini and W G Pitt Colloids

Surf B 55 59 (2007)67 G A Husseini N M Abdel-Jabbar F S Mjalli and W G Pitt

Technol Cancer Res Treatment 6 49 (2007)68 J Pruitt Stabilization of Pluronic P-105 for Targeted Nanoparti-

cle Drug Delivery PhD Dissertation Brigham Young UniversityProvo Utah (2001)

69 A Chattopadhyay and E London Anal Biochem 139 408 (1984)70 N Rapoport and Z Gao Polymer Reprints 47 49 (2006)71 N Rapoport Prog Polym Sci 32 962 (2007)72 N Rapoport Z G Gao and A Kennedy J National Cancer Insti-

tute 99 1095 (2007)73 G A Husseini R I El-Fayoumi K L OrsquoNeill N Y Rapoport

and W G Pitt Cancer Letters 154 211 (2000)74 G A Husseini K L OrsquoNeill and W G Pitt Technol Cancer Res

Treatment 4 707 (2005)75 J D Pruitt and W G Pitt Drug Delivery 9 253 (2002)76 A Marin M Muniruzzaman and N Rapoport J Controlled

Release 75 69 (2001)77 A Marin H Sun G A Husseini W G Pitt D A Christensen

and N Y Rapoport J Controlled Release 84 39 (2002)78 A Marin M Muniruzzaman and N Rapoport J Controlled

Release 71 239 (2001)79 N Rapoport A Marin Y Luo G D Prestwich and

M Munirzzaman J Pharm Sci 91 157 (2002)80 M D Muniruzzaman A Marin Y Luo G D Prestwich W G Pitt

G Husseini and N Y Rapoport Colloids Surf B Biointerfaces25 233 (2002)

81 G A Husseini C M Runyan and W G Pitt BMC Cancer 2 1(2002)

82 H H Lin and Y L Cheng Macromolecules 34 3710 (2001)83 H J Lin P Herman J S Kang and J R Lakowicz Anal Biochem

294 118 (2001)84 N Rapoport Int J Pharm 227 155 (2004)85 N Sheikov N McDannold F Jolesz Y Z Zhang K Tam

and K Hynynen Ultrasound in Medicine and Biology 32 1399(2006)

86 S B Stringham B K Murray K L OrsquoNeill S Ohmine T AGaufin and W G Pitt 96th Annual Meeting of the AmericanAssociation for Cancer Research 1415 AACR Anaheim CA USA(2005)

87 M R Bailey L N Couret O A Sapozhnikov V A KhokhlovaG Ter Haar S Vaezy X G Shi R Martin and L A Crum Ultra-sound in Medicine and Biology 27 695 (2001)

88 E S Richardson W G Pitt and D J Woodbury Biophys J 934100 (2007)

2214 J Nanosci Nanotechnol 8 2205ndash2215 2008

Delivered by Ingenta toAmerican Scientific Publishers

IP 6923324200Sat 07 Jul 2012 184331

REVIEW

Husseini and Pitt The Use of Ultrasound and Micelles in Cancer Treatment

89 B Howard A Gao S-W Lee M-H Seo and N Rapoport Am JDrug Deliv 4 97 (2006)

90 R K Schlicher H Radhakrishna T P Tolentino R P ApkarianV Zarnitsyn and M R Prausnitz Ultrasound in Medicine andBiology 32 915 (2006)

91 B J Staples Pharmacokinetics of Ultrasonically-Released Micelle-Encapsulated Doxorubicin in the Rat Model and its Effect onTumor Growth MS Thesis Brigham Young University Provo UT(2007)

92 J L Nelson Ultrasonically Enhanced Drug Delivery of DoxorubicinIn Vivo from Stabilized Pluronic Micelle Carriers MS BrighamYoung University Provo UT (2002)

93 J L Nelson B L Roeder J C Carmen F Roloff and W G PittCancer Res 62 7280 (2002)

94 Z G Gao H D Fain and N Rapoport J Controlled Release 102203 (2005)

95 Z G Gao D H Lee D I Kim and Y H Bae J Drug Targeting13 391 (2005)

96 G Myhr and J Moan Cancer Lett 232 206 (2006)97 M D Lucroy Compendium 24 140 (2002)98 Z Gao H D Fain and N Rapoport Molecular Pharmaceutics 1

317 (2004)99 N Rapoport D A Christensen H D Fain L Barrows and Z Gao

Ultrasonics 42 943 (2004)

Received 16 July 2007 Accepted 6 August 2007

J Nanosci Nanotechnol 8 2205ndash2215 2008 2215