Ultrasound in Med. & Biol., Vol. 39, No. 12, pp. 2342–2350, 2013Copyright � 2013 World Federation for Ultrasound in Medicine & Biology

Printed in the USA. All rights reserved0301-5629/$ - see front matter

/j.ultrasmedbio.2013.06.002

http://dx.doi.org/10.1016

d Original Contribution

NON-INVASIVE MAGNETIC RESONANCE IMAGING FOLLOW-UP OFSONO-SENSITIVE LIPOSOME TUMORDELIVERYANDCONTROLLEDRELEASE

AFTER HIGH-INTENSITY FOCUSED ULTRASOUND

ROBERT ANDREW FOWLER,*yz SIGRID L. FOSSHEIM,x JEAN-LOUIS MESTAS,*y JACQUELINE NGO,*y

EMMANUELLE CANET-SOULAS,yz and CYRIL LAFON*y

*LabTAU, Inserm, U1032, Lyon, France; yUniversit�e de Lyon, Lyon, France; zCarMeN, Inserm, U1060, Lyon, France; andxEpitarget AS, Lysaker, Norway

(Received 26 July 2012; revised 5 April 2013; in final form 6 June 2013)

Aserm ULyon C

Abstract—This work examines the use of lanthanide-based contrast agents and magnetic resonanceimaging in monitoring liposomal behavior in vivo. Dysprosium (Dy) and gadolinium (Gd) chelates, Dy-diethylenetriaminepentaacetic acid bismethylamide (Dy-DTPA-BMA) and Gd-DTPA-BMA, were encapsulatedin pegylated distearoylphosphatidylethanolamine-based (saturated) liposomes, and then intravenously injectedinto Copenhagen rats with subcutaneous Dunning AT2 xenografts. Liposome-encapsulated Dy chelate shortenstransverse relaxation times (T2 and T2*) of tissue; thus, liposomal accumulation in the tumor can be monitoredby observing the decrease in T2* relaxation time over time. The tumor was treated at the time of maximumliposomal accumulation (48 h) with confocal, cavitating high-intensity focused ultrasound to induce liposomalpayload release. Using liposome-encapsulated Gd chelate at high enough concentrations and saturated lipo-somal phospholipids induces an exchange-limited longitudinal (T1) relaxation when the liposomes are intact;when the liposomes are released, exchange limitation is relieved, thus allowing in vivo observation of payloadrelease as a decrease in tumor T1. (E-mail: andrew.fowler@inserm.fr) � 2013 World Federation for Ultra-sound in Medicine & Biology.

Key Words: Cavitation, Magnetic resonance imaging, Liposomes, High-intensity focused ultrasound.

INTRODUCTION

Liposomes have been studied as vectors for gene therapy,carriers of chemotherapeutic drugs and transporter mole-cules used for diagnosis and imaging (Barenholz 2001;Kim and Kwon 2010; Lammers et al. 2010). Of themany potential applications for liposomes, drug-loadedliposomes have found a niche in tumor treatment.Because of the well-documented enhanced permeabilityand retention (EPR) effect (Maeda 2010), smaller dosesof chemotherapeutic drugs can be administered, andthus the damaging side effects of chemotherapy are alsoreduced. Briefly, the EPR effect is caused by the leakyvasculature found in tumors, which allows nanoscaleparticles circulating within the vascular system to leakout into the tumor. It is thought that the triggered releaseof a chemotherapeutic payload from liposomes after

ddress correspondence to: Robert Andrew Fowler, LabTAU, In-1032, Batiment INSERM, 151 Cours Albert Thomas, 69424edex 03, France. E-mail: andrew.fowler@inserm.fr

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a phase of circulation and accumulation in the tumorwill increase therapeutic efficacy even further. High-intensity focused ultrasound (HIFU) and liposomalrelease have seen much development both with drug-loaded liposomes and with liposomes loaded withimaging agents (Dromi et al. 2007; Myonopoulou et al.2010; Negussie et al. 2011; Smit et al. 2010; Staruchet al. 2011). Many treatments that combine HIFU andliposomal release rely largely on thermal releasemechanisms and use ultrasound as a targeted, in vivoheat source (Cornelis et al. 2011; Wang et al. 2010).The HIFU approach suggested for this application,however, is non-thermal, and relies on the mechanicaleffects of inertial cavitation to trigger liposomal release(Chen and Wu 2010; Evjen et al. 2012; McMaughlanet al. 2010; Somaglino et al. 2011).

A tandem drug delivery scheme is easy to conceiveand test in vitro, but it is difficult to ascertain the behaviorof drug carriers in vivo, especially release induced byultrasound. Biodistribution of liposomes in a relativelyshort time frame after injection has been observed using

MRI follow-up of liposome delivery/release after HIFU d R. A. FOWLER et al. 2343

18F-labeled liposomes with positron emission tomography(Tartis et al. 2008). Tartis et al. provided key insight intothe fate of the lipid components in liposomes after in-jection, but were not able to show the behavior of theliposomal payload and, thus, no indication of release.Conversely, many techniques for evaluating efficacy andbioavailability of the liposomal payload, such as confocalmicroscopy and flow cytometry, require animal sacrificeor ex vivo experimental conditions (Biswas et al. 2012;Hallow et al. 2007). Other analytical techniques, such asatomic emission spectroscopy, mass spectrometry andquantitative nuclear magnetic resonance spectroscopy,require, in addition, separation techniques such as sizeexclusion chromatography and high-performance liquidchromatography to assess liposomal accumulation andrelease (Paulis et al. 2012; Schroeder et al. 2009; Skidanet al. 2011). Given the recent advances in magneticresonance-guided HIFU, a non-invasive method forobserving the fate of liposomes and their associatedpayload in vivo with magnetic resonance imaging (MRI)would be useful.

The aim of this study was to observe liposomebehavior and tumor accumulation in vivo, as well as lipo-some interaction with therapeutic ultrasound, validatingthe in vitro data generated from the liposome formulationstudies. The liposomes chosen for this study were smalland were formulated for long circulation times tomaximize the EPR effect (Gabizon et al. 1994; Hagtvetet al. 2011; Lafon et al. 2012; Evjen et al. 2013). Asa result of the physicochemical properties (i.e., notthermally sensitive) of this formulation, non-thermalHIFU was chosen as the triggering mechanism for lipo-somal release andwas applied at the time of maximal lipo-some accumulation in the tumor. To visualize liposomalbehavior, the liposomes were loaded with two differentMRI contrast agents, chosen to illustrate liposomal accu-mulation and payload release, respectively. To visualizeaccumulation, dysprosium diethylenetriaminepentaaceticacid bismethylamide (Dy-DTPA-BMA, sprodiamide)was used. Dy-DTPA-BMA has a magnetic susceptibilityor T2* effect if compartmentalized within a liposome,for instance, and, for this reason, was chosen as a T2* orsusceptibility contrast agent to follow liposome deliveryand accumulation in the tumor (Castelli et al. 2009;Fossheim et al. 1997). The Gd chelate, Gd-DTPA-BMA(Gadodiamide), displays a markedly different T1 relaxiv-ity when encapsulated in rigid liposomes (often composedof saturated phospholipids) at a sufficiently high enoughconcentration compared with free in solution, and forthis reason, it was chosen to visualize liposomal release(Burtea et al. 2004; Fossheim et al. 1998; 1999; 2000;Strijkers et al. 2005). To verify these hypothesesregarding the proposed relaxation effect of theencapsulated contrast agents, the actual T1 and T2*

relaxivities of intact and solubilized liposomes weremeasured in vitro. When these contrast agents are usedin combination, liposomal accumulation in tumor tissueshould be observable as a decrease in T2* or T2relaxation time, and liposomal release on exposure toHIFU should be observable as a decrease in T1relaxation time concomitant with increased signalintensity on T1-weighted MR images.

METHODS

Liposome preparation and characterizationLiposomes were prepared by Epitarget AS (Lysaker,

Norway) and contained a 62:8:30 molar ratio of distear-oylphosphatidylethanolamine (DSPE), DSPE with2000-Da polyethylene glycol moiety (DSPE-PEG2000),and cholesterol, respectively (Evjen et al. 2010). Phos-pholipids were purchased from Genzyme Pharmaceuti-cals (Liestal Switzerland), and cholesterol waspurchased from Sigma-Aldrich (St. Louis, MO, USA).Liposomes were made by the thin-film hydration method(Lasic 1993). Lipids were dissolved in a mixture of chlo-roform and methanol (9:12, v/v) at 60�C, and dried ina rotary evaporator in a vacuum for 1 h at 70�C to forma thin lipid film. The thin film was then hydrated 74�Cwith a solution of 125 mM Gd DTPA-BMA, 125 mMDy DTPA-BMA and 10 mM calcein (as a fluorescenceindicator molecule for the progress of dialysis duringthe manufacturing process). Liposomes were allowed toswell for 2 h at 74�C before being subjected to threefreeze-thaw cycles using a methanol/acetone bath cooledwith dry ice. The liposomes were then brought to a diam-eter below 90 nm using sequential extrusion through twostacked polycarbonate membranes with varying porediameter (from 800 to 80 nm) at 74�C (Lipex Extruder,Biomembrane, Vancouver, BC, Canada; Nucleporefilters, West Chester PA, USA). After extrusion, excesscalcein and lanthanide contrast agents were removed bydialysis against an isotonic solution of sucrose containing10 mMHepes buffer (pH 7.4). A sample from the batch tobe used in the study was sent for ICP-AES (inductivelycoupled plasma atomic emission spectroscopy) quantifi-cation of lanthanide concentration in the liposome disper-sion (performed by the analytical company Eurofins,Moss, Norway). Concentrations were measured at 1.90mM Gd and 1.86 mM Dy; measurements were repeateduntil a sufficiently low confidence interval had beenreached. The estimated final lipid concentration was16 mg/mL. The final liposome size after dialysis was86 nm as measured by photon correlation spectroscopy(Zetasizer, Malvern Instruments, and Worcestershire,UK). The polydispersity index was measured as 0.080indicating a very narrow size distribution.

2344 Ultrasound in Medicine and Biology Volume 39, Number 12, 2013

In vitro liposomal relaxation properties were mea-sured at 7 T in a small-bore magnet (Billerica, MA, USA)by placing 500-mL aliquots of liposome dispersion (non-diluted and diluted with 10 mM Hepes sucrose solution)into Eppendorf tubes held in a 37�C aqueous phantom.Measurements were taken for 53, 23 and neat dilutions,with three repetitions for each point. Complete contrastagent release from the liposomes was induced by the addi-tion of 10 mL 10% Triton-X detergent (purchased fromSigma-Aldrich),mixed in avortexmixer, and 5minheatingina 65�Cwater bath.T1 relaxivity coefficients for intact andsolubilized liposomes were calculated from a saturationrecovery sequence, and T2* relaxivity coefficients werecalculated from images acquired using gradient echo-pulse sequences. The corresponding liposomal T1 andT2* relaxivities were then calculated by linear regressionanalysis of T1 and T2* relaxation rates versus Gd and Dyconcentrations, respectively.

Magnetic resonance imaging

Image acquisition. Experiments were run on a 7-Tsmall-bore magnet (Bruker, Billerica, MA, USA). Theanimals were placed under isoflurane anesthesia withcardiac and respiratory monitoring. Image acquisitionwas performed with the animals in the prone position,with localization achieved using an initial single-sliceFLASH sequence for coronal, sagittal and axial posi-tioning. The tumoral protrusion was used as an anatomiclandmark for positioning before and after HIFU. Theimaging lasted approximately 45 min. The probe usedfor emission and reception was a Bruker T10720 V3linear volume coil (300 MHz, inner diameter 5 72 mm,outer diameter 5 120 mm).

The pulse sequence used for T1 measurements wasa fast spin echo sequence, that is, RARE (rapid acquisi-tion with relaxation enhancement), with variable relaxa-tion time (VTR) for T1 relaxometric measurements. Thesequence used 18 repetition times from 245 to 4315 ms.Two-dimensional images were acquired: 10 contiguousslices, matrix size 128 3 128. X and Y resolutionswere both set at 0.47 mm, and slice thickness was set at1 mm. For T2* measurements, a 2-D multi-echo gradientecho (MGE) was used, with the following parameters:20 contiguous slices 25 echoes from 25 to 697 ms with28-ms echo spacing, 2563 256 matrix, spatial resolutionof 0.234 mm in both directions, and slice thickness of1 mm. Before application of the MGE sequence,a magnetic field map was taken to account for T2* varia-tion caused by field inhomogeneities (Abduljalil andRobitaille 1999; Volz et al. 2009).

Image analysis. Parameter maps were created withMRIUtil, developed by the Center for NMR Researchat Penn State University Hershey College of Medicine

(University Park, PA, USA). T1 maps were created usingthe three-parameter saturation recovery model

AðB2e^2ðTR=T1ÞÞwhere A and B are coefficients solved for by optimization,and TR is the repetition time from the VTR sequence. T2*maps were created using the non-linear two-parametermodel

Ae^2ðTE=T2�Þwhere A is solved for using optimization, and TE is theecho time given by the MGE sequence. Relaxation valuesfor the tumor were averaged by drawing a region ofinterest for the tumor on each slice of the parameter mapin ImageJ, and averaging the value across the entire tumor(National Institutes of Health, Bethesda, MD, USA).

In vivo modelThese animal experiments were approved by an

independent ethics committee and are in agreementwith the national ethical laws for animal experimentation.

Tumor implantation was performed at the Animal-erie Lyon-Est Conventionnelle (ALECS), Facult�e de Me-decine RTH Laennec, Lyon, France, and ultrasound andMR experiments were performed at the Grenoble Institutdes Neurosciences, Grenoble, France. Male Copenhagenrats (Charles River Laboratories, L’Arbresle, France)with a subcutaneously implanted cubic 25-mg fragmentof the AT2 phenotype of Dunning R3327 (LGC standards,Molsheim, France) prostatic adenocarcinoma were usedfor the experiments (Fizazi and Navone 2005). Accordingto previous work, this tumor is susceptible to the EPReffect when using doxorubicin-loaded liposomes (Lafonet al 2012). Also, the Dunning tumor model has beenfound to react synergistically with high-intensity ultra-sound combined with chemotherapeutics (Paparel et al.2005). For each step of the experiment (tumor implanta-tion, ultrasound treatment and MRI), all animals wereunder gaseous anesthesia (Isoflurane [Belamont], Nicho-las Piramal, UK). Two studies were performed on 13rats: one study acquired images every 24 h over the courseof 72 h to observe liposomal accumulation in the tumor,and the other acquired images at 0 and 48 h and focusedon liposomal release. Only rats in the liposomal releasestudy were exposed to ultrasound. Each study had twogroups: those injected with liposomes (n 5 6 amongboth studies), and those that received no injection (n 57 among both studies). As there was overlap betweenthe two studies with respect to experimental conditionsand imaging sequences acquired, where possible, ratsfrom both studies were included in the statistical analysis.As treatment with ultrasound destroys the liposomes or, atthe very least, causes some release, observation of

MRI follow-up of liposome delivery/release after HIFU d R. A. FOWLER et al. 2345

liposomal accumulation can no longer be performed withgroups that have received ultrasound treatment. To avoidconfusion, the number of animals analyzed at each datapoint is indicated in each figure. The lipid dosage admin-istered was chosen to have the same amount of liposomesthat would be injected in a protocol using similar-sizedliposomal doxorubicin for a chemotherapeutic evaluationin rats; thus, injection was determined by lipid concentra-tion. This valuewas set at 96mg/kg for lipid dose, with thevolume injected per rat �1.5 mL. Calculating from thisvolume, the contrast agent dosage was 11 mmol/kgdysprosium and 11 mmol/kg gadolinium. These areconsiderably lower doses than the clinically recommen-ded Gadodiamide dose of 50–100 mmol/kg for centralnervous system applications (Food and Drug Administra-tion Prescribing Information Ref: 2881213).

UltrasoundFigure 1 is a diagram of the ultrasound setup. The rat

was placed on its side in a plastic bed with a window cut

Fig. 1. Diagram of high-intensity focused ultrasound deviceand experimental setup. The animal was placed in a bed underisofluorane anesthesia. Instrumentation: Arbitrary waveformgenerator (AM Gen), Oscilloscope (Osc), 1kW RF Amplifier(1kW), Wattmeter (W). The tumor was localized with B-modeechographic imaging, and its geometry was demarcated witha MATLAB (Mathworks, Natick, MA, USA) script. The tumorwas held exposed with an elastic. The tumor was then sweptthrough the focal zone according to the diagram in Figure 2.

for the tumor. The tumor area was exposed by shaving thefur and applying depilatory cream. The subcutaneoustumor was tied with a string to enhance the protrusionfrom the skin. Acoustic coupling was achieved bylowering the bed with the rat into a tank of degassed waterheld at 37�C. The ultrasonic device consisted of two iden-tical transducers placed in a confocal orientation, with thetwo beams crossed at an 110� angle, improving the spatialresolution of treatment. The transducers operated ata frequency of 1.1 MHz. Pulsed emission was performedwith an Agilent 33120 A arbitrary waveform generator(Agilent Technologies, Santa Clara CA, USA) at a pulserepetition frequency of 250 Hz and pulse length of 40 ms.Acoustic pressure was measured using a calibratedM€uller-Platte needle hydrophone (M€uller Ingenieurtech-nik, Bensheim, Germany) and a LeCroy Wavesurfer 422oscilloscope (LeCroy, Chestnut Ridge NY, USA). Peaknegative pressure at the focal point was found to be25.7 MPa, and peak positive pressure was 46.2 MPa.Focal diameter was measured to be 2 mm in the lateraland elevational axes and 3 mm in the axial axis. Cavita-tion was observed by B-mode imaging during treatmentwith a Hawk 2102 ultrasound scanner using a 12-MHzlinear imaging probe (BK Medical, Herlev, Denmark).The presence of transient hyperechogenic regions duringtreatment were taken as an indication of cavitation. Addi-tionally, broadband noise observed in the backscatteredsignal measured from the transducers lends additionalevidence of the presence of inertial cavitation. Datafrom this observation were previously published inSomaglino et al. (2011). The rat tumor was swept throughthe focal zone along the axial axis at 1 mm/s, with 2 mmbetween each plane, using an ESP300 motor controller(Newport, Irvine CA, USA). The entire volume of thetumor was treated. Positioning automation was doneusing in-house scripts written with the MATLAB instru-ment control toolbox (Mathworks, Natick, MA, USA).Figure 2 is a diagram of the treatment sweep path.

Statistical analysisResults are expressed as the mean 6 standard devi-

ation; coefficients of variation were used to express theevolution of inhomogeneities within the region ofinterest. Changes in relaxation coefficients were calcu-lated by subtracting average values for the entire tumorvolume. Percentage changes were also calculated withmean values for the entire volume. T1 and T2* resultswere verified using the analysis of variance single-factor test. As liposome accumulation was inhomoge-neous, analysis of T1 was performed on zones of maximalliposomal accumulation, taking care to keep a consistenttumor size over the course of the follow-up. Analysis wasperformed using MATLAB and the Excel 2010 DataAnalysis Pack (Microsoft, Redmond WA, USA).

Fig. 2. Diagram of sweep path. The tumor was localized with a B-mode imaging probe. After location and demarcation,the tumor was swept through the focal zone according to the diagram at a speed of 1 mm/s with 2 mm between each plane.This sweep speed and geometry mean that all locations within the demarcated zone of the tumor spend 2 s within the

ultrasonic focus.

2346 Ultrasound in Medicine and Biology Volume 39, Number 12, 2013

RESULTS

In vitro relaxivity characterizationTo confirm that longitudinal and transverse relaxiv-

ities did indeed differ between intact and solubilized lipo-somes, T2* and T1 relaxivities were measured in vitro.Three serial dilutions of intact and lysed liposomeswere made, and relaxivity was calculated by least-squares fitting. Standard deviations for relaxation timesfor all dilutions were observed to be less than 1% of themean. For intact liposomes, T2* relaxivity was 0.079 s-1

mM-1 Dy (R2 5 0.9991). Solubilized liposomes hada T2* relaxivity of 0.012 s-1 mM-1 Dy (R2 5 0.9673).The reduced T2* relaxivity after liposome solubilizationindicates a lesser extent of Dy compartmentalization. T1relaxivities of 1.09 s-1 mM-1 Gd (R2 5 0.9259) and5.04 s-1 mM-1 Gd (R2 5 0.9994) were obtained for intactand solubilized liposomes. The T1 relaxivity values forsolubilized liposomes (mimicking Gd-DTPA-BMArelease) reported here are slightly higher than previouslyreported for Gd-DTPA-BMA free in solution at similarhigh field strength and 37�C (Caravan 2006; Kellaret al. 1997). This could be due to imperfect thermalcoupling, as a temperature lower than 37�C in thephantom would correspond to a higher relaxivity forreleased Gd-DTPA-BMA. Overall, however, these relax-ation data indicate that the liposome formulation exhibitssuitable properties to ascertain in vivo liposome accumu-lation and HIFU-mediated release in the tumor.

In vivo imaging of liposomal accumulation and releaseThe rats were selected at random from a group of 13,

yielding 3 rats per group in each study, with an extra ratassigned to the group receiving no injection in the accu-mulation study. One rat in the liposomal injection groupfrom the accumulation study died from anesthesiacomplications at 48 h. Mean tumor size during thefollow-up was 1.68 6 0.35 cm3 (n 5 13) at 48 h (timeof ultrasound treatment). Liposomal accumulation was

observed using T2* mapping. Figure 3 is a sample T2*map of the subcutaneous tumor over a 72-h period afterinjection of liposomes. On average, 14 of the 20 sliceswere used for calculations at each time point. It can beseen from Figure 3 that there is a nadir in T2* values 48h after injection. Figure 4 illustrates the average changein T2* values measured for the entire tumor volume ateach time point. The average change in T2* in the tumorat the 48-h time point was26.496 2.68 ms for rats (n56,230% relative to day 0 baseline T2*) that had been in-jected with liposomes, compared with 11.07 6 2.19 ms(n 5 4, 16% relative to day 0 baseline T2*) for thenon-injected animals (p , 0.002 at the nadir of 48 h).Liposomal accumulation within the tumor was nothomogenous, and an effect was not observable uniformlyin all slices. Values given in the figures represent averagestaken for the entire tumor volume. Peak changes in T2*were observed at the interface of the leg muscle and thetumor.

In Figure 5 are T1 maps before and after treatmentwith ultrasound. On average, 8 of 10 slices were used.The average change in T1 over the tumor volume withsignificant T2* change was2212.76 70.7 ms, comparedwith the 25.2 6 26.9 ms observed for the control(p 5 0.009) (Fig. 6).

DISCUSSION

The goal of this study was to ascertain the accumu-lation of liposomes in a tumor model and then to observethe release of the payload on exposure to ultrasound. Thehypotheses that many liposomal formulations do in factaccumulate in a tumor after injection and that this partic-ular formulation will release its payload when exposed toHIFU treatment were posed. The data gathered from thisstudy support both the accumulation and release hypoth-eses. Given that such a system lives up to its claims, thiswould have both implications for the further use of thiscombination of lanthanide contrast agents and for the

Fig. 3. Sample T2* maps from the accumulation study with time scale in milliseconds. The top row is injected with lipo-somes, and the bottom is the non-injected control group. It can be seen that T2* relaxation time, as represented by imageintensity, reaches a nadir 48 h after injection in the group that received the injection (decrease of 26% in this example).

MRI follow-up of liposome delivery/release after HIFU d R. A. FOWLER et al. 2347

development of this liposomal formulation in particular.This method also is an improvement on existing methodsthat require animal sacrifice and destructive analyticalchemistry techniques (Paulis et al. 2012; Schroederet al. 2009; Skidan et al. 2011), in that it allowslongitudinal study at time points after treatment withinthe same animal. The application of this technique islargely pre-clinical, though it is a useful first step in eval-uating the in vivo behavior of drug-loaded nanoparticles

Fig. 4. Comparison of variations in T2* in the accumulationstudy between injected rats and non-injected rats (control).DT2* is expressed relative to average T2* measured for thetumor at the time of injection. T2* relaxation time reaches itsnadir 48 h after injection. n 5 number of animals analyzed ateach time point. It can be seen that there is a significant differ-ence at 48 h (p , 0.002), which is not present at any other time

point.

designed for triggered release. Given the large interestin encapsulating a variety of drugs for cancer and otherclinical indications, this technique can be used to ensurethe stability and controlled release of liposome formula-tions and other nanoparticles in vivo.

The T2* relaxivity measured in vitro was higher forintact versus solubilized liposomes from which the Dychelate is released. As T2* effects are greatly reducedfor solubilized liposomes, it is likely that the T2* effectthat is observed in vivo with this tumor model is indeeddue to intact liposomal accumulation. The large standarddeviation seen at 72 h can be at least partially attributed tonecrosis in the core of the tumor. This necrosis can beseen in anatomic T2 images and is part of the tumorprogression in this model.

The T1 relaxivity of Gd-DTPA-BMA is significantlylower when encapsulated in liposomes than when in freesolution, as is observed by relaxivity measurements re-ported here. Given the previous in vitro characterizationwork with this formulation and its release on exposureto ultrasound (Evjen et al. 2010; Hagtvet et al. 2011),and the agreement between the proposed mechanismfor liposomal Gd-based MR contrast presented here andin vitro studies performed, it is reasonable to concludethat there is release from liposomes on treatment withultrasound with several caveats. As the T1 relaxivityof liposomal Gd-DTPA-BMA is dependent on waterexchange between the liposome interior and exterior(bulk), it is possible that the reduction in T1 that is

Fig. 5. Sample T1 maps from the release study, with time scale in seconds. Top: animals injected with liposomes, bottom:non-injected animals (control). Left: before ultrasound (US), right: after US. T1 values are represented by image intensity.It can be seen that there is a significant drop in T1 values after treatment as compared with control animals. T1 inhomo-geneity increases after treatment (pixel-to-pixel coefficient of variation increases from 7.6% to 10.2% in the controlanimal), and tumor size increases slightly, but the average T1 relaxation time changes very little (10.3 ms in this case).

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observed in the tumor represents merely a permeabiliza-tion of the liposomes after ultrasound treatment, allowingwater to rapidly cross the membrane, but not allowing the

Fig. 6. Comparison of variations in T1 in the release studybetween the injected group and the non-injected (control) group(n5 3 for both groups), with time scale in milliseconds. Samplesize (n) is indicated.DT1 is calculated for each tumor as the differ-ence in the average T1 value for the whole tumor before and aftertreatment. As can be seen, there is a significant differencebetween the ultrasound-treated, liposome-injected group andthe ultrasound-treated non-injected (control) group (p5 0.009).

liposomal payload into the tissue. This question cannot beanswered definitively with the current protocol and data,but in vitro release of doxorubicin and calcein (water-soluble molecules of about same molecular weight asthe Gd chelate) with this liposomal formulation and othersimilar formulations, on exposure to cavitating ultra-sound, has been reported (Evjen et al. 2010; Evjen et al.2011; Hagtvet et al. 2011; Somaglino 2011). Also,the standard deviation of the T1 relaxation time wasindeed large (coefficient of variance 5 33%), but aspreviously mentioned, liposomal accumulation was nothomogenous. Lastly, there is indeed a large T1 effect inthe absence of ultrasound when comparing T1 at 0 and48 h in rats that have and have not been injected withliposomes, with a change of 2345.9 6 86.6 ms (p ,0.002, shown in supplemental data). This indicates thatliposomes have a significant effect on T1 before the appli-cation of ultrasound. The effect of liposomal accumula-tion on tumor T1 in the absence of ultrasound suggeststwo non-exclusive possibilities. First, the water exchangeacross the lipid bilayer is indeed significant, thus, so is therelaxation effect of intact liposomes. This is corroborated

MRI follow-up of liposome delivery/release after HIFU d R. A. FOWLER et al. 2349

by the non-zero relaxivity of intact liposomes andmust betaken into account when applying this method to otherliposome formulations. Second, the payload may be leak-ing from the liposomes to a certain extent and thus isbeing released before the application of ultrasound.Additional experiments coupling size exclusion chroma-tography and ICP-AES could be used to quantify theactual extent of liposomal payload release.

Both the liposomal formulation and the contrastagent combination at high intra-liposomal concentrationpresent opportunities for future work. The high concen-tration of cholesterol in this liposomal formulation makesit ideal for release studies using MRI contrast agentswhich are dependent on interaction with bulk water.However, depending on the intra-liposomal concentrationof Gd-based contrast agent used, it may be less feasible touse this type of paramagnetic contrast agent with lipo-somes that have higher water membrane exchange rates,such as liposomes with low cholesterol levels or liposomeformulations composed of unsaturated lipids (Methaiet al. 2008).

With respect to the utility of the contrast agentcombination, these two contrast agents can be used forevaluation of other liposomal formulations in vivo. Thatsaid, this technique is an alternative for experimentsthat aim to evaluate the accumulation and release of lipo-somal payloads in vivo using destructive methods such asthose mentioned in the Introduction. Additionally, Dy-and Gd-based contrast agents have the advantage of beingnon-biologically occurring nuclei, allowing analysis ofin vivo accumulation using atomic quantification tech-niques such as atomic emission spectroscopy. The disad-vantage to using such contrast agent molecules is thattheir physicochemical properties are not closely relatedto those of many molecules used in tumor therapy and,therefore, cannot be used directly to determine drug avail-ability in the tumor. In future work with this technique,we will apply the findings here to other liposomal formu-lations to evaluate their potential for tumor-targeted trig-gered delivery.

CONCLUSIONS

This study explored the in vivo behavior of long-circulating DSPE-based liposomes. Liposomal accumu-lation could be illustrated using MRI, thus proving thelong-circulating nature of this formulation. Though quan-titative assessment of liposomal release requires furtherwork, and perhaps a more uniformly developing tumormodel, the images acquired in this study indicated thatonce liposomes are accumulated, liposomal release isinduced after treatment with ultrasound. These resultsare significant in two ways: the peak accumulation at48 h (as shown with T2* relaxation measurements) indi-

cates the stability and long circulation time of this lipo-somal formulation in vivo, and the efficacy of HIFU ininducing release in such a formulation is determinedusing T1 relaxation measurements. This lays experi-mental groundwork for further use of this liposomalformulation in delivering drug payloads to other biologictargets and indicates the efficacy of using cavitation toinduce release.

Acknowledgments—This Eureka labeled project (E!4056) is supportedby the French Norwegian Foundation and OSEO, and performed withinthe framework of the LABEX DEVweCAN (ANR-10-LABX-0061) ofUniversit�e de Lyon, within the program ‘‘Investissements d’Avenir’’(ANR-11-IDEX-0007) operated by the French National ResearchAgency (ANR). The authors thank Sibylla Røgnvaldsson for the supplyof liposomes, Chantal R�emy, Emmanuel Barbier, Vasile Stupar, RegineFarion, and Cl�ement Debacker from the Imaging platform of the Greno-ble Institute of Neurosciences for help with MRI data acquisition.Thanks to GE Healthcare for providing Dy-DPTA-BMA. The develop-ment of MRIUtil is supported in part through NIH R01 EB006563, R01AG02771 and the Pennsylvania Department of Health.

SUPPLEMENTARY DATASupplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ultrasmedbio.2013.06.002.

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