Assessment of Intratumoral Doxorubicin Penetration after ...described in Derieppe et al. [23]. Brieﬂy, this automated pipeline includes cell detection, which was facilitated by a

Research ArticleAssessment of Intratumoral Doxorubicin Penetration afterMild Hyperthermia-Mediated Release fromThermosensitive Liposomes

Marc Derieppe 1 Jean-Michel Escore1 Baudouin Denis de Senneville12

Quincy van Houtum1 Angelique Barten-van Rijbroek1 Kim van der Wur-Jacobs1

Ludwig Dubois3 Clemens Bos 1 and Chrit Moonen1

1Imaging Division University Medical Center Utrecht Utrecht Netherlands2Institut de Mathematiques de Bordeaux UMR 5251 CNRS Universite de Bordeaux Bordeaux France3Maastro Lab Maastricht University Maastricht Netherlands

Correspondence should be addressed to Marc Derieppe mppderieppe-3prinsesmaximacentrumnl

Received 5 December 2018 Accepted 14 February 2019 Published 7 March 2019

Guest Editor Igor Nabiev

Copyright copy 2019 Marc Derieppe et al shyis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In solid tumors rapid local intravascular release of anticancer agents eg doxorubicin (DOX) from thermosensitive liposomes(TSLs) can be an option to overcome poor extravasation of drug nanocarriers shye driving force of DOX penetration is the drugconcentration gradient between the vascular compartment and the tumor interstitium In this feasibility study we used beredconfocal uorescence microscopy (FCFM) to monitor in real-time DOX penetration in the interstitium of a subcutaneous tumorafter its intravascular release from TSLs shyermodoxreg Cell uptake kinetics of the released DOX was quantied along with an in-depth assessment of released-DOX penetration using an evolution model A subcutaneous rat R1 rhabdomyosarcoma xenograftwas usedshye rodent was positioned in a setup including a water bath and FCFM identication of functional vessels in the tumortissue was applied based on AngioSense shye tumor-bearing leg was immersed in the 43degC water for preheating and TSLs wereinjected intravenously Real-time monitoring of intratumoral (it) DOX penetration could be performed and it showed theprogressing DOX wave front via its native uorescence labeling successively all cell nuclei Cell uptake rates (1k) of 3 minuteswere found (n 241 cells) and a released-DOX penetration in the range of 2500 microm2middotsminus1 was found in the tumor extravascularspaceshyis study also showed that not all vessels identied as functional based on AngioSense gave rise to local DOX penetration

1 Introduction

Local drug delivery strategies in oncology aim at increasingdelivery of anticancer drugs in the tumor while limitingtheir exposure in healthy tissues that induces toxic sideeects eg cardiotoxicity of the small molecule doxorubicin(DOX relative molecular mass of 544Da) [1 2] Onestrategy devised in previous studies is the systemic injectionof long-circulating nanomedicine formulations such aspegylated liposomes to facilitate drug accumulation in thetumor tissue [3 4] shyis can be achieved by exploiting twopathophysiological characteristics of solid tumors that

constitute the enhanced permeability and retention eect(EPR) [5 6] (1) a higher vascular permeability because of alack of vessel dierentiation and (2) insucentcient functionallymphatics Despite this passive drug targeting penetrationof nanoparticles into the tumor interstitium is rather limited[7 8] and variable according to the tumor type [3]Moreover nanoparticles display slow drug release in thetumor tissue which often results in drug accumulationbelow the minimum concentration required to induce celldeath [9]

shye advent of temperature-sensitive liposomes (TSLs)with the rst formulation described by Yatvin et al [10]

HindawiContrast Media amp Molecular ImagingVolume 2019 Article ID 2645928 13 pageshttpsdoiorg10115520192645928

allowed the design of new drug delivery strategies with a localtriggering by an external source of mild hyperthermia -esedrug delivery systems must fulfill four main criteria (1) a longplasma half-life (2) a stable formulation at body temperature(3) a phase transition temperature corresponding to aphysiologically mild and safe hyperthermia hence the nameof low temperature-sensitive liposomes (LTSLs) and (4) aldquofastrdquo drug release rate from the liposomes when more than50 of their payload was released within around half a minutewhen exposed to their phase transition temperature [11 12]Needham and coworkers were the first to incorporate lyso-lipids in themembrane bilayer and paved the way for an LTSLformulation that was clinically compatible [13]

Combination of LTSLs with an external heat sourceallowed local treatment and Manzoor et al introduced theconcept of triggered rapid intravascular release [14] uponexternal triggering the LTSLs release the drug payloadrapidly in the tumor vasculature and build up a drugconcentration gradient between the tumor vasculature andthe tumor tissue which constitutes the driving force ofdiffusion that is the main source of transport for smallmolecules eg DOX [15] -is drug penetration is pro-portional to the drug concentration gradient Using TSLswith a fast drug release rate one can therefore maximizedrug penetration into the tumor

To conduct drug distribution studies at the tissue scaledorsal skin-fold window chambers have been used in vivo-ese consist of a skin flap held vertically with an aluminiumsaddle that is sutured to the upper and the lower parts of theflap [16ndash18] An optical window gives access to the fascialtissue and its vasculature and tumor cells can be implantedinto this window chamber An alternative intravital imagingsolution is fibered confocal fluorescence microscopy(FCFM) this modality allows real-time fluorescence imag-ing in a minimally invasive fashion by directly placing afiber-based optical probe in contact with the tissue of interestin its natural physiological environment FCFM is mainlyused in clinical applications involving hollow organs like ingastroenterology [19] and pulmonology [20] either visu-alizing the tissue based on its autofluorescence or afterinjection of fluorescein or indocyanine green

In this study we tested the feasibility to use FCFM tomonitor in vivo in real-time DOX penetration in the tumorinterstitium after intravascular release of DOX from the TSL(-ermodoxreg) -e kinetic analysis from the time seriesallowed quantifying (1) the local uptake kinetics of releasedDOX in each individual cell of the interstitium after releasefrom the TSL (2) the kinetics of the apparent released-DOXpenetration using the transport equation and (3) thereleased-DOX deposition the vascular washout and thedrug diffusion by means of an evolution model from thefluorescence signal intensity

2 Materials and Methods

21 Experimental Setup

211 Animals and Tumor Model All procedures wereperformed according to the ethical guidelines and were

approved by the animal welfare committee of UtrechtUniversity (DEC 2014III03035 Utrecht the Netherlands)WAGRij rats were purchased from Charles River (CologneGermany) -ey were maintained at room temperature with12 h light cycle in individually ventilated isolation cages andwere fed ad libitum -e rats were 12weeks old at the be-ginning of the experiments weighing 250 g Under gaseousanesthesia (Aerrane Baxter Deerfield IL) a skin incision ofa few millimeters was performed at the hind leg Sub-sequently rat R1 rhabdomyosarcoma tumor pieces (1ndash3mm3) were subcutaneously implanted in the hind leg usinga trocar When the tumor volume reached 1500 microm3 ap-proximately after 3weeks of tumor growth the drug ad-ministration and imaging experiments were performed

212 Chemicals Lysothermosensitive liposomal formula-tion of DOX (-ermodoxreg-TSL) at 2mgmL was obtainedfrom Celsion Corp (Lawrenceville NJ USA) -ese nano-particles release their payload as a burst in the temperaturerange of 395degC to 42degC ie less than 5 of release at 37degCand more than 65 at 41degC within about 30 sec (in vitro dataprovided by Celsion Corp) On the day of the real-timemonitoring experiment the -ermodoxreg solution was fil-tered using a PD10-desalting column (GE HealthcareEurope GmbH Eindhoven the Netherlands) to ensure thatthe DOX penetration that was monitored was fully encap-sulated previously in the TSL -e rodents were adminis-tered intravenously with a -ermodoxreg dose of 4mgkg

Doxorubicin hydrochloride (Sigma-Aldrich St-LouisMO) (relative molecular mass 580Da) named ldquofreeDOXrdquo in this study was injected intravenously at 4mgkg

An intravascular fluorescence label AngioSense 680 EXwas purchased from Perkin Elmer (Waltham MA USA)AngioSense is a 70 kDa near-infrared labeled-fluorescentpolymer (excitationemission wavelengths 670690 nm)which allows imaging the blood pool during the wholeimaging session

213 Fibered Confocal Fluorescence MicroscopyFluorescence images were acquired in real-time (85Hz) for20minutes using a dual-band FCFM system (Cellvizioregdual-band Mauna Kea Technologies Paris France) Nativefluorescence of DOX was collected with the 488 nm exci-tation channel henceforth referred to as ldquogreen channelrdquoand blood vessels via AngioSense with the 660 nm channelreferred to as ldquored channelrdquo -eir spectral sensitivity is500ndash630 nm and 680ndash800 nm respectively A 15 mm di-ameter FCFM microprobe (PF-2210 Mauna Kea Technol-ogies) was used (Figure 1(c)) with the following imagingspecifications lateral resolution 33 μm field of view (FOV)602 μmtimes 602 μm axial resolution 15 μm working distance0 μm-e tissue was excited at 4mW of laser power for bothchannels

214 Animal Positioning A water bath (MemmertSchwabach Germany) was used to ensure a mild and ho-mogeneous tumor heating (Figure 1(b)) -e water

2 Contrast Media amp Molecular Imaging

temperature was monitored using ber optic temperaturesensors (Luxtron Lumasense Technologies GmbH Frank-furt Germany) A custom-made platform was designed toposition the animal above the water surface and to immerseonly the tumor bearing leg (Figure 1(b))

shye animal was thermally isolated from the water bathusing a piece of aluminium foil (Figure 1(b)) shye rectaltemperature and the water temperature (set to 43degC) in thevicinity of the tumor were monitored during the sessionDuring the whole experiment the rectal temperature waslower than the phase transition temperature of the TSL(Figure S1) shye hind leg bearing the tumor was positionedin the water bath for 10minutes which was sucentcient to getthe tumor tissue at 43degC (data not shown)

Handling of the FCFMmicroprobe was facilitated by usinga modular hose holding it in xed position in contact with thetumor tissue such that a dynamic microscopy time series of axed tumor location could be obtained (Figure 1(b))

215 Timeline of the Imaging Session shye rats wereanaesthetized with an ip injection of 75mgkg of ketamine(Narketan Vetoquinol rsquos-Hertogenbosch the Netherlands)

and 025mgkg of dexmedetomidine (Dexdormitor OrionPharma Mechelen Belgium) shyen the jugular vein wascatheterized a 1 cm skin ap was created at the tumor level(Figure 1(a)) and the rats were then injected intravenouslywith 192 nmolmiddotkgminus1 of AngioSense (Figure 1(d))

Subsequently the rats were positioned on the platform ofthe water bath laying on the ank with the tumor bearing legimmersed in the 43degC water Temperature probes were thenplaced to monitor the temperature of the water bath and thebody temperature of the animal shye tip of the FCFM probewas placed manually to make contact with the tumor tissue inthe water and the tumor was explored until tortuous tumorvessels were found shyen stability of the FOV was veried bywaiting one minute and by checking any motion of the tumormicrovasculature in the image shye syringe containingshyermodoxreg was only then placed in the catheter to avoidpremature heating of the liposomes At this point the 20-minute real-time monitoring was started After collecting the10-second baseline with tissue autouorescence a 4mgkgbolus injection of TSLs or free DOX was administered in-travenously in the jugular vein in around 40 s

After completion of the dynamic sequence explorationof the tumor surface was performed manually with the

(a) (b)

5 mm

(c)

Anesthesia

10 s 50 s

Experimental Timeline

Jugular veincatheterization

Skin flap fortumor access

AngioSense injection

Rat positioningin setup

Search fortumor vasculature

ThermoDoxreg injection(jugular vein)

Tumor preheating Dynamic monitoring

Tumor screeningwith FCFM probe

Organ blood andurine harvesting

(d)

Figure 1 Tumor access (a) setup (b) tip of the FCFM probe (c) and timeline of the experiment (d) After incision of the skin at the tumorlocation (a) the rat was positioned on a platform placed at the water surface of the water bath with the hind leg immersed in the water set to43degC (b)

Contrast Media amp Molecular Imaging 3

FCFM probe andmicrographs were acquired to evaluate thepresence of DOX in the tumor microenvironment by meansof its native fluorescence [21 22]

At the end of the session the rats were sacrificed by anip injection of 200mgkg of pentobarbital (Euthanimal20 AlfasanWoerden the Netherlands) and the blood theurine if any and the tumor were harvested

Two treatment groups consisted of the ldquofree DOXrdquo group(n 3) and the ldquoTSLrdquo group (n 5)

3 Histopathology and Liquid Biopsy Analysis

Tumors were harvested and fixed in the formol-acetic acidsolution -en histological samples were embedded inparaffin cut at approximately 5 μm and prepared usingconventional hematoxylineosin protocol -e tissue sec-tions were examined by light microscopy on a Keyencemicroscope (Keyence International Belgium) Whole tumorimaging was performed using a 4x magnification objective(numerical aperture 06) and the mosaicking module with a7-by-7 matrix -e size of the neoplastic nodule on the slidewas measured with a ruler-en micrographs of each tumorarea (necrotic versus proliferative areas) were acquired at20x magnification using a PlanFluor objective (Numericalaperture 05) and at 60x using an oil-immersion PlanApoVC objective (numerical aperture 14)

Micrographs of DOX fluorescence in frozen-tissuesections were acquired using a Leica TCS SP8 X confocalfluorescence microscope with a 10x magnification objectivea 504 nm excitation wavelength and an emission filter of540ndash680 nm

DOX concentration in blood and urine samples wasmeasured using Ultra Performance Liquid Chromatography(UPLC) with fluorescence and UV detection An ACQUITYUPLC BEH C18 separation column was used (130 Angstromsize of 17micron 17times 50mm) -e mobile phase consistedof an eluant with the following mixture 75 reverse-osmosiswater 25 acetonitrile and 1 perchloric acid Duringseparation a column temperature of 50degC and a sampletemperature of 25degC were set with 3 minutes of run time-efluorescence was measured with a 480 nm excitation565 nmemission and a UV detection of 234 nm

4 Kinetic Analysis of Released-DOX Penetration

-e real-time fluorescence image data obtained was pro-cessed offline using MATLABreg 2013 (-e MathWorksNatick MA USA) To increase the signal-to-noise ratio thesequence was averaged temporally to an 8 s frame rateKinetic analysis of DOX penetration consisted of the 3following independent but complementary sections

41 Cell Uptake Kinetics of Released-DOX Cell uptake ki-netics was assessed using the dedicated parametric pipelinedescribed in Derieppe et al [23] Briefly this automatedpipeline includes cell detection which was facilitated by anonlocal means algorithm [24] a cell tracking with the

iterative-closest-point algorithm [25] and a fitting of theresulting uptake profiles by means of a two-compartmentmodel where the fluorescence signal intensity (I) is as follows[26]

I(t) A 1minus eminusk(tminusT)

1113960 1113961 (1)

where A is the maximum DOX fluorescence signal T thetime of signal onset and k the uptake rate -e model wasconsidered accurate when Pearsonrsquos correlation coefficient(r2) was greater than 095 the values of the uptake rate ofreleased DOX were reported as median with interquartileranges -en a statistical analysis of the resulting phar-macokinetic parameters of the cell population visible in theFOV was performed

42 Analysis of the Released-DOX Penetration in the TumorInterstitium Quantitative evaluation of released-DOXpenetration in the tumor interstitium was then performedby estimating the instantaneous apparent DOX transport(noted V

rarr) using the transport model applied to all acquired

images [27] In practice the following equation was appliedin a homogeneous environment to calculate the in-stantaneous released-DOX penetration between timepoints tand t+ δt

It + Vrarr

middot nablararr

I 0 (2)

where I denotes the native fluorescence signal monitored inthe green channel and It the partial temporal derivative of Icalculated between the timepoints t and t+ δt-e left part ofthis equation consists of a transient term (It) and an ap-parent transport V

rarrmiddot nablararr

I which stand for any temporal andspatial grey intensity variations respectively -e sum ofboth terms equal 0 to ensure the signal intensity conser-vation with motion in the field of view Any spatiotemporalintensity variations occurring between timepoints t andt+ δtmay be attributed to DOX transport in the model used-e estimated transport field V

rarrthus accounts for spatio-

temporal fluorescence intensity variation occurring duringthe dynamic imaging sequence

-e transport model of equation (2) is intrinsicallyunderdetermined thus leading to an ill-conditioned nu-merical scheme -e transport field V

rarrwas therefore com-

puted on a pixel-by-pixel basis by applying the minimizationprocess as follows

argminVrarr

1113946Ω

1113868111386811138681113868It + Vrarr

middot nablararr

I1113868111386811138681113868 + α1113874

nablararr

u2

2 +nablararr

v2

21113875 d rrarr

(3)

where ΩsubeR2 is the coordinate domain of the image (u v)the components of transport vectors estimated and r

rarr isin Ωthe spatial location -e minimized functional consists ofboth additive contributions as follows (1) a fidelity term (leftpart of the integral in equation (3)) that optimizes throughan L1 norm the transport model of equation (2) given thatan L1 penalizer is applied transient variations occur iden-tically and regardless of the grey level intensity (2) a reg-ularization term (equation (3) right part of the integral)designed to introduce a sufficient conditioning to the


numerical scheme -e regularization term is given bynablararr

u2

2 u2x + u2

y andnablararr

v2

2 v 2x + v 2

y ux uy vx with vy

being the spatial partial derivatives of u and v respectivelyPhysically this regularization term assumes that thetransport between neighboring pixels is moderate

-e data fidelity and the regularization terms are linked bythe weighting factor α that was set to 10 in the scope of thisstudy-is value was motivated by the fact that a high α valueincreases the stability of the numerical scheme but alsohampers in turns the ldquospatial elasticityrdquo of the estimateddisplacement field In order to optimize the computation timeand to ensure the convergence of the algorithm a multi-resolution scheme that iterated the registration algorithm wasused from a four-fold down-sampled image step-by-step tothe full image resolution [28]-e interested reader is referredto Corpetti et al [29] for more information about the nu-merical resolution of dense estimation of fluid flows

In order to mitigate the local impact of the nucleusfluorescence signal on DOX penetration in the interstitiuma spatial low-pass Butterworth filter was applied to eachindividual image before the resolution of equation (3) Inorder to remove cell nuclei with typical diameters up to10 μm according to an image pixel size of 10times10 microm2 thecut-off frequency fc of the low-pass filter was set tofc f016 f0 being the original image sampling frequency

-e onset of fluorescence signal was defined when themaximum fluorescence signal in the current image exceededat least 5 of the maximum signal of the sequence Since thetransport model of equation (2) relies on the spatiotemporalfluorescence intensity the analysis of the released-DOXpenetration in the interstitium was performed on imagesacquired after this timepoint

A principal component analysis (PCA) was appliedsubsequently on the time series of the computed 2-di-mension penetration vector fields in order to find the spatialorthogonal basis the resulting principal axis served togenerate an adequate representation of the sequence ofdisplacement fields An averaged motion amplitude alongeach principal axis was then calculated for each individualdisplacement field and allowed calculating the temporalprofile of the average motion amplitude along each principalaxis

43 Modeling of Released-DOX Penetration Ultimately thespatiotemporal distribution of released-DOX fluorescencesignal intensity S was modeled and included a uniformreleased-DOX deposition δ a vascular washout ω pro-portional to the current fluorescence signal intensity and ahomogeneous released-DOX apparent diffusion ] in theFOV with the following equation

z

ztS( r

rarr t) δ minusω middot V( r

rarr) middot S( r

rarr t) + ] middot ΔS( r

rarr t) (4)

where Δ is the Laplacian operator t is the time instant rrarr

(x y z) is the voxel coordinate and V( rrarr

) is a probabilitydensity function (PDF) that has a value close to 1 (0 re-spectively) in voxels having a high (low respectively)probability to be in the vascular space Here only diffusion

was considered as diffusion is dominant for small mole-cules such as doxorubicin in the extravascular space [15]

In this model the fluorescence signal intensity S( rrarr

t)

was collected in the green channel -e PDF V( rrarr

) wasderived from the fluorescence signal in the vessels collected inthe red channel at the beginning of the acquisition-is signalwas normalized between 0 and 1 and assumed to be tem-porally invariant (a linear relation between the fluorescencesignal in the vessels and the PDF was assumed to be a goodapproximation in the scope of this study) -e coefficients δω and ] were assumed to be spatially and temporally in-variant within a temporal window covering the time periodstarting from 100 seconds which corresponds to the onset offluorescence signal in the FOV to 20minutes of imaging

Equation (4) was solved using a finite difference methodintegrated in an explicit Euler scheme in order to simulatethe evolution of fluorescence signal intensity for specificvalues of δ ω and ] Using a LevenbergndashMarquardt fit thecoefficients δ ω and ] were calculated to minimize the least-square residue between the model and the measured dataover the complete sequence -e algorithm applied multipleregularly sampled initial conditions for ] in order to preventthe algorithm from falling into local minima -e goodnessof the fit as evaluated by Pearsonrsquos correlation coefficientwas computed in order to evaluate whether the model de-scribed accurately the fluorescence signal enhancement

Since cell nuclei are nonmoving structures the impact oftheir fluorescence signal was reduced using a spatial low-pass Butterworth filter (order 1) in each individual imagebefore the numerical resolution of equation (4) Similar tosection 42 the cut-off frequency fc of the low-pass filter wasset to fc f016

5 Results

51 Histopathological Analysis To evaluate the influence ofmild hyperthermia on tumor tissue histopathological an-alyses were carried out at the end of our imaging sessionTumors were visible as rounded neoplastic nodules presentwithin the subcutaneous and muscle tissues and partiallysurrounded by thin fibrous capsules All tumors (37degC vs43degC) exhibited common histopathological characteristics-e proliferation areas weremade up of poorly differentiatedneoplastic cells within a fine vascular stroma -ese cellswere highly pleomorphic with one or more prominentnuclei Some multinucleated cells were also observed in alltumors -e transition proliferativenecrotic areas are well-defined with a loss in cell density and a clear fibrillary aspectthat is characteristic of the extracellular matrix in the ne-crotic area (25ndash30 of all tumor nodules) Few multifocalinfiltrations of neutrophils were observed in all tumors Noapparent tissue damage in any of the areas exposed to a 43degClocal hyperthermia (Figure S2) was observed in comparisonto the control tumors (Figure S2)

52 Real-Time Monitoring of Released-DOX PenetrationIn the red channel staining of the blood pool by AngioSenseallowed finding functional characteristically tortuous


tumor microvasculature (Figures 2(a) 2(c) 2(e) and 2(g))In the free DOX group iv injection did not lead to a de-tectable FCFM signal (n 3) neither during the real-timemonitoring (Figure S3) nor during histopathology(Table S1) Conversely in the TSL group (n 5) a fluo-rescence signal enhancement was collected (n 2 Table S1)but not in the other animals (n 3) Due to image drift inone animal only the onset of DOX fluorescence increasecould be analyzed but not the complete DOX dynamics anddistribution

-e fluorescence signal enhancement was present in theextravascular space as well as in the cell nuclei of the tumorinterstitium which reflected intracellular uptake of releasedDOX Interestingly the onset of DOX uptake in the cellsstarted from one side and progressively spread to involvecells throughout the FOV (Figures 2(b) 2(d) 2(f) 2(h) and2(i)) However no DOX fluorescence enhancement wasvisible in vessels In the red channel the fluorescence ofblood vessels decreased during the acquisition probablyowing to photobleaching but it was still possible to locatethem until 12minutes of acquisition indicating that theposition of the FOV was steady

53 Cell-Uptake Kinetics of Released-DOX Real-timemonitoring of released-DOX penetration in the tumorinterstitium shows cell nuclei that display increasing fluo-rescence signal (Figures 2(b) 2(d) 2(f) 2(h) and 2(i)) thusindicating an increasing DOX concentration upon celluptake 241 cell nuclei could be detected and were includedin the analysis -e maximum fluorescence intensity derivedfrom the fit (124 au with interquartile range of 49 au)(Figure 3(a)) did not display any spatial correlations in themaximum-fluorescence-intensity map neither in the FOVnor with the position of the vessels in the FOV (Figure 3(b))Of all nuclei the median of uptake rates 1k was 2minutes54 s (interquartile range 4minutes 40 s) (Figure 3(d))however remarkably faster uptakes (range 0ndash3minutes 20 s)were observed in the upper-right half of the FOV(Figure 3(e)) In this case no clear spatial correlation withthe microvasculature could be identified (Figure 3(e) reddotted lines in background) indicating that released-DOXinducing the fluorescence signal did not originate from themicrovessels visible in the FOV but instead from vesselsbeyond the confocal slice

54 Analysis of the Interstitial Released-DOX Penetration-e spatiotemporal distribution of the released-DOX nativefluorescence directly reflected released-DOX penetration(Figures 4(a)ndash4(e)) and was assessed using the fluid dy-namics model described by equation (2) through the min-imization process of equation (3) -e arrival of DOX wasvisible in the upper-right corner of the FOV 3minutes afterbolus injection and pointed toward the lower-left corner ofthe FOV (Figure 4(i))

-e principal component analysis allowed determiningthe main direction of released-DOX penetration with 94of relative displacement along the principal axis 1(Figure 4(k)) -e temporal profile of relative displacement

along the principal axis 1 displayed a periodic pattern(Figure 4(l)) Released-DOX penetration then equilibratedin the FOV 4minutes after the detected fluorescence onsetaccording to the low-amplitude displacement vectors(Figure 4(j))

55 Modeling of Released-DOX Penetration Released-DOXpenetration was modeled using the evolution model ofequation (4) (Figure 5)-e quality of the fit as computed bythe sample Pearsonrsquos correlation coefficient was equal to089 reflecting a close match of our proposed model with theexperimental data It is to be noticed that this model onlyused the background fluorescence as input with a negligibleimpact of the nucleus fluorescence observed in the acquiredsequence (Figures 5(a)ndash5(d)) this was confirmed by themaps of the residuals (Figures 5(i)ndash5(l)) ie the differencebetween the experimental data and the evolution model -eestimated drug diffusion ] was approximately 2500 microm2middotsminus1(see Figure 5(m))

56 Observation of the Tumor Surface by FCFM Aftermonitoring dynamically DOX penetration after in-travascular release at a single location manual exploration ofthe tumor surface by the FCFM probe showed heterogeneityof DOX distribution in the tumor (Table S1) Interestinglythe native fluorescence of DOX was predominantly locatedclose to the vessels imaged in the red channel (Figure S4a)and showed a pattern similar to that found during the real-time monitoring with high signal from the cell nuclei(Figure S4b) However despite the presence of tortuousvessels (Figure S5a) a nonnegligible amount of locations inthe same tumor did not show any DOX fluorescence(Figure S5b) suggesting heterogeneity in DOX distributionin the tumor In these cases after sacrifice of the animal atumor incision allowed insertion of the FCFM probe forscreening of the tumor tissue and also showed high DOXheterogeneity within the tumor tissue (data not shown) thisheterogeneity was confirmed in DOX fluorescence micro-graphs collected from ex vivo samples (Figure S6)

6 Discussion

Evaluation of drug penetration in the tumor microenvi-ronment is key to optimize therapeutic strategies ofhyperthermia-triggered drug delivery in solid tumors In thisfeasibility study a setup was devised to monitor DOXpenetration in the tumor interstitium in real time afterintravascular release of DOX from TSL Here we usedFCFM as an alternative of commonly used dorsal skinfoldwindow chambers to assess drug penetration in amechanically unrestricted tumor microenvironment

First no apparent microstructural changes could beobserved in tumor tissue exposed to a 43degC mild hyper-thermia thus confirming the noninvasive nature of thehyperthermia procedure Real-time imaging using FCFMwas performed at the tumor surface with a skin incision andallowed imaging separately the vascular compartment andreleased DOX after intravascular release from the TSL


In the TSL group the real-time monitoring of DOX wassuccessful in 2 out of 5 cases in the TSL group only but DOXwas found in every tumor when exploring the tumor rim atthe end of the imaging session In the free DOX group noDOX could be detected neither during the real-timemonitoring nor during the tumor exploration at the endof the imaging session suggesting that the DOX

concentration was too low to be detected in the extravascularspace shyis could be explained by a low concentrationgradient between the tumor vasculature and the extravas-cular space thus limiting free DOX penetration hence theuse of TSLs

Released-DOX penetration kinetics could be assessedupon imaging of cell-uptake kinetics of released DOX in the

0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)

12 min(g) 12 min (h)

Green channelRed channel

0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)

Figure 2 Real-time monitoring of doxorubicin penetration in the tissue microenvironment after TSL intravascular release (a c e g) Redchannel with AngioSenseTM (blood-pool labeling) showed that the acquisition was performed in a steady FOV and that the vessels werefunctional (b d f h i) shye green channel shows DOX uorescence signal enhancement after the bolus injection of TSL


Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001

Map of maximum fluorescence intensity (au)

(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200

Acquisition time (min)

Fluo

resc

ence

sign

al in

tens

ity (a

u)

Experimental dataFit 1k = 3 min 17 s (r2 = 0985)

Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200

Map of uptake rates 1k (min)

min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2

Figure 3 Uptake rates 1k derived from the two-compartment model Boxplots of the maximum DOX uorescence intensity (a) and theuptake rate 1k (d) and their corresponding spatial distributions respectively (b e) Characteristic examples of uorescence signal en-hancements collected in two nuclei (dark plain curves) and the tted curves derived from the two-compartment model (dashed red curves)(c f ) Two distinct subpopulations of uptake rates in the FOV (e) are signicantly dierent (g)


tumor interstitium and DOX diusion coecentcient both inthe same dataset Here 241 cell nuclei could be detectedusing the image processing pipeline developed previously[23] thus allowing a statistical analysis Uptake time con-stants of 3minutes were found after injection of 4 mgkgDOX encapsulated in TSLs Interestingly no apparent linkcould be made between the location of the microvasculatureand the direction of the propagation front when observingcell uptake of DOX in the FOV shye uptake rates around3minutes show faster uptakes than those found in previousstudies where uptake rates 1k around 10minutes werefound with free DOX [30 31] shyis would conrm theusefulness of TSLs where a TSL intravascular release in-duces higher DOX levels in the extravascular space and thushigher DOX concentration gradients as a driving force forthe faster cell uptake

Using the transport model of equation (2) together witha principal component analysis the released-DOX pene-tration in the presented results was shown to occur mainlyalong one axis oriented from the top-right to the bottom-left

corner of the FOV (Figure 4(k)) Relative DOX displacementwas found to equilibrate 7minutes after the detected DOXarrival in the FOV shyis nding would mean that theconcentration gradient which is the driving force for DOXpenetration into the tumor interstitium was not presentanymore between the vascular compartment and theinterstitium after 7minutes shye estimated relative dis-placements here indicate that like in the cell uptake kineticsof released-DOX computed above no spatial correlationbetween the displacement eld and the location of themicrovasculature was found In the scope of this study it isimportant to underline that an apparent displacement iscalculated here without taking the uid rheology into ac-count shyis will be worth considering in future studies

It is important to underline that any spatiotemporalintensity variations occurring between timepoints t andt+ δt in equation (2) may be attributed in our model toldquodisplacementrdquo shyis assumption brings a limitation withregards to released-DOX penetration in the interstitiumwhich is likely to be biased by the uorescence signal in the

Disp

lace

men

t fie

lds

DO

X flu

ores

cenc

e3 min 10 s 3 min 40 s 4 min 10 s 5 min 20 s 13 min 10 s

0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

Principal component analysis (PCA)

0 5 10 15 20

Relative displacement along eigenvector 1

Time of acquisition (min)

Detected onset of fluorescence in FOV

minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)

Figure 4 Assessment of released-DOX penetration derived from the implemented uid dynamics model (andashe) Fluorescence imagesmeasured at 3min 10 s (a) 3min 40 s (b) 4min 10 s (c) 5min 20 s (d) and 13min 10 s (e) (fndashj) Corresponding released-DOX penetrationshown by the displacement eld For an easier visualization only displacement vectors associated to voxels with sucentcient uorescencesignal (ie greater than 5 of the maximum uorescence signal intensity) are displayed shye principal component analysis alloweddetermining the main direction of released-DOX penetration with 94 of relative displacement along the eigenvector 1 (k) shye temporalprole of relative displacements along the eigenvector 1 equilibrated 7minutes after the uorescence onset (l)


0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)

4 min 8 min 12 min 16 min

1000 2000 3000 4000 5000

0052

0053

0054

0055

0056

Diffusion (μm2middotsndash1)

Resid

ue o

f the

leas

t-squ

are f

it (a

u) Drug diffusion estimate

(m)

Drug deposition estimate

1000 2000 3000 4000 5000Diffusion (μm2middotsndash1)

Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)


Vascular washout estimate

Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150

Figure 5 Modeling of released-DOX penetration in the tumor microenvironmentshye implemented evolution model allowed calculating adrug diusion of ] 2500 microm2middotsminus1 (endashh) Only the background uorescence served in the evolution model as shown by the absolutedierence maps (indashl) calculated between the experimental data and the computed released-DOX penetration by the evolution model shyeparameters to apply were calculated for the released-DOX penetration model (ie equation (4))shye x-axis reports regularly sampled valuesof the diusion parameter ] that are exhaustively enumerated during the optimization process (m) shye optimization of the released-DOXapparent diusion parameter ] the averaged least-square residue between the model and the measured data was minimal for] 2500 microm2middotsminus1 Optimal released-DOX deposition δ and vascular washout ω are displayed for each value of ] in (n) and (o) respectively


cell nuclei as these are nonmoving image structures Oursimulations were thus conducted on spatially filtered fluo-rescence images in order to circumvent this issue -ereforeit is to be noted that the apparent DOX velocity estimateslogically increased when a reduced cut-off frequency fc wasapplied in the low-pass spatial-image filtering process be-cause in this case nuclei weighted less in filtered imagesUsing fc f016 and fc f08 DOX velocity estimatesdisplayed less than 10 of variations thus confirming thatthe local impact of the fluorescence signal in the cell nucleiwas properly discarded

-e diffusion coefficient reflects the magnitude of drivingforce generated by the concentration gradient built up be-tween the vascular compartment and the tumor interstitium-e model of released-DOX penetration yielded a diffusionestimate around 2500 microm2middotsminus1 In the present study anothersource of uncertainty arose from the linear intensity of thevessel likelihood that was assumed to be located along the redfluorescence signal intensity (equation (4)) Consequently asmall but nonnegligible drug evacuation is also assumed in theinterstitium where positive nonzero values are found in theimages in the red channel It is important to report that forinstance a greater diffusion coefficient would logically befound if no evacuation in the interstitium is assumed in ourmodel Nonetheless this diffusion estimate is greater than the40 microm2middotsminus1 reported in previous studies using free DOX[9 30] or 150 microm2middotsminus1 found by Swabb et al who establishedthe correlation between the tissue diffusion coefficient at 37degCand the solute molecular weight ie 544Da for DOX [32]-is one-order-of-magnitude discrepancy may be in line withthe use of TSLs because the payload released locally yieldshigher DOX concentration in the vasculature thus increasingDOX concentration gradient between the vasculature and theextravascular space and in turn a higher diffusivity

Another source of bias in the evaluation of the diffusioncoefficients may here arise from the 2D observation of a 3Ddiffusion process this 2D observation is due to the design ofthe FCFM Hence only a 2D implementation of equation (4)could be applied that neglects out-of-plane drug diffusionGiven that the FCFM probe used here allowed imaging from0 to 15 μm of depth this bias could only come from out-of-plane events occurring deeper than the 15 μm of confocalslice

After monitoring dynamically DOX penetration at asingle location the manual exploration of the tumor surfaceand its depth by the FCFM probe showed a clear spatialheterogeneity in DOX native fluorescence despite thepresence of tortuous vessels and the homogeneous heatingensured by the water bath

Despite the detection of functional vessels with Angio-Sense no real-time DOX penetration could be observed byFCFM in 3 out of the 5 animals in the TSL group whereashistopathology showed high DOX concentration in the tumorarea in all animals (n 5 Table S1) In addition as clearlyshown in Figure 2 DOX penetration does not arise from allfunctional vessels Vessel functionality is identified in thisstudy as showing AngioSense fluorescence It should be notedthat AngioSense has been injected well before the TSL in-jection and that its blood half-time is long (around 7h data of

supplier) Taken together these data indicate that vessels withAngioSense-based functionality are not all functional withrespect to DOX penetration upon TSL injection Two possiblemechanisms could explain this either TSL did not (yet) arrivein the FCFM observed region or the TSL already lost theirDOX payload upstream before arrival -e latter explanationmay be relevant because of the very fast release of DOX fromTSL upon hyperthermia (on the order of seconds) and shouldbe considered when combining hyperthermia and TSLs forregional drug delivery

Our FCFM and data processing approach could alsoserve for the study of extravasation of drug nanocarrierseg its enhancement by hyperthermia [33] provided thatthe nanocarriers can be sufficiently fluorescently labeled Inthis study however we followed the tumor preheatingprotocol in order to maximize drug uptake in the tumorwhich leads to immediate intravascular release from theliposomes [34] It is noteworthy that this approach may leadto a better understanding of the relation between drugpenetration and tumor physiology characteristics such asthe interstitial fluid pressure [35] and the solid stress de-scribed recently [36] Last the image processing frameworkconducted in this study can be used in a broad range ofexperiments where imaging modalities at the tissue scaleallow collecting pharmacokinetic parameters for the eval-uation of drug formulations and drug nanocarriers [37]

7 Conclusions

FCFM can provide real-time visualization of DOX in vivo ina mechanically unrestricted tumor microenvironmentallowing the study of DOX penetration at the tissue scale-e proposed image analysis framework provides data oncell uptake kinetics as well as penetration kinetics of releasedDOX and thus helped to characterize the driving forceconstituted by the concentration gradient after intravascularrelease of DOX from the TSL

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that they have no conflict of interest todisclose

Acknowledgments

-is study was funded by the ERC project 268906 ldquoSoundPharmardquo (Prof C Moonen) -e authors are grateful toBurcin Ozbakir (Pharmaceutical Sciences Institute Uni-versity of Utrecht Utrecht the Netherlands) and DrNoboru Sasaki (Image Sciences Institute University MedicalCenter Utrecht Utrecht the Netherlands) for their technicalsupport -e authors also thank Prof Gert Storm for fruitfuldiscussions (Pharmaceutical Sciences Institute University ofUtrecht Utrecht the Netherlands)


Supplementary Materials

Table S1 summary of the results rat-by-rat DOX concen-trations in urine and blood correspond to the results ofsingle quantitative measurements Figure S1 representativeprofiles of rat rectal temperature recorded during real-timemonitoring Figure S2 micrographs showing the impact ofmild hyperthermia on the tumor microstructure in theproliferative area (b c i j) the transition area (d e k l) andthe necrotic area (f g m n) of the rat 4 No morphologicaldamage was observed at 43degC (hndashn) compared to 37C (andashg)in any of the 3 areas Figure S3 real-time monitoring ofdoxorubicin penetration in the tissue microenvironmentafter free DOX intravenous injection (a c e g) Red channelwith AngioSenseTM (blood-pool labeling) showed that theacquisition was performed in a steady FOV (b d f h) NoDOX fluorescence signal enhancement could be observed inthe green channel after free DOX intravenous injectionFigure S4 micrographs collected after dynamic monitoringIn the interstitium nuclei that take up doxorubicin (b) arepredominantly located around the vessels (a) Figure S5micrographs acquired after dynamic monitoring Despitethe presence of tortuous vessels characteristic of tumortissue (a) a nonnegligible amount of areas in the tumortissue did not show any doxorubicin signal (b) Figure S6representative fluorescence micrographs (mosaicking) of atumor tissue exposed to TSL DOX distribution is hetero-geneous Objective 10x (Supplementary Materials)

References

[1] E T H Yeh D J Lenihan S W Yusuf et al ldquoCardiovascularcomplications of cancer therapyrdquo Circulation vol 109 no 25pp 3122ndash3131 2004

[2] D Outomuro D R Grana F Azzato and J MileildquoAdriamycin-induced myocardial toxicity new solutions foran old problemrdquo International Journal of Cardiologyvol 117 no 1 pp 6ndash15 2007

[3] K J Harrington S Mohammadtaghi P S Uster et alldquoEffective targeting of solid tumors in patients with locallyadvanced cancers by radiolabeled pegylated liposomesrdquoClinical Cancer Research vol 7 no 2 pp 243ndash254 2001

[4] T Lammers F Kiessling W E Hennink and G StormldquoDrug targeting to tumors principles pitfalls and (pre-)clinical progressrdquo Journal of Controlled Release vol 161 no 2pp 175ndash187 2012

[5] Y Matsumura and H Maeda ldquoA new concept for macro-molecular therapeutics in cancer chemotherapy mechanismof tumoritropic accumulation of proteins and the antitumoragent Smancsrdquo Cancer Research vol 46 no 12 pp 6387ndash6392 1986

[6] H Maeda J Wu T Sawa Y Matsumura and K HorildquoTumor vascular permeability and the EPR effect in macro-molecular therapeutics a reviewrdquo Journal of Controlled Re-lease vol 65 no 1-2 pp 271ndash284 2000

[7] G Kong R D Braun and MW Dewhirst ldquoCharacterizationof the effect of hyperthermia on nanoparticle extravasationfrom tumor vasculaturerdquo Cancer Research vol 61 no 7pp 3027ndash3032 2001

[8] M R Dreher W Liu C R Michelich M W DewhirstF Yuan and A Chilkoti ldquoTumor vascular permeabilityaccumulation and penetration of macromolecular drug

carriersrdquo JNCI Journal of the National Cancer Institutevol 98 no 5 pp 335ndash344 2006

[9] A W El-Kareh and T W Secomb ldquoA mathematical modelfor comparison of bolus injection continuous infusion andliposomal delivery of doxorubicin to tumor cellsrdquo Neoplasiavol 2 no 4 pp 325ndash338 2000

[10] M Yatvin J Weinstein W Dennis and R BlumenthalldquoDesign of liposomes for enhanced local release of drugs byhyperthermiardquo Science vol 202 no 4374 pp 1290ndash12931978

[11] B Kneidl M Peller L Lindner G Winter and M Hossannldquo-ermosensitive liposomal drug delivery systems state of theart reviewrdquo International Journal of Nanomedicine vol 9no 1 pp 4387ndash4398 2014

[12] D Needham J-Y Park A MWright and J Tong ldquoMaterialscharacterization of the low temperature sensitive liposome(LTSL) effects of the lipid composition (lysolipid and DSPE-PEG2000) on the thermal transition and release of doxoru-bicinrdquo Faraday Discussvol 161 no 0 pp 515ndash534 2013

[13] D Needham G Anyarambhatla G Kong andM W Dewhirst ldquoA new temperature-sensitive liposome foruse with mild hyperthermia characterization and testing in ahuman tumor xenograft modelrdquo Cancer Research vol 60no 5 pp 1197ndash1201 2000

[14] A A Manzoor L H Lindner C D Landon et al ldquoOver-coming limitations in nanoparticle drug delivery triggeredintravascular release to improve drug penetration into tu-morsrdquo Cancer Research vol 72 no 21 pp 5566ndash5575 2012

[15] M W Dewhirst and T W Secomb ldquoTransport of drugs fromblood vessels to tumour tissuerdquo Nature Reviews Cancervol 17 no 12 pp 738ndash750 2017

[16] H D Papenfuss J F Gross M Intaglietta and F A TreeseldquoA transparent access chamber for the rat dorsal skin foldrdquoMicrovascular Research vol 18 no 3 pp 311ndash318 1979

[17] S Hak N K Reitan O Haraldseth and C de Lange DaviesldquoIntravital microscopy in window chambers a unique tool tostudy tumor angiogenesis and delivery of nanoparticlesrdquoAngiogenesis vol 13 no 2 pp 113ndash130 2010

[18] L Li T L M ten Hagen D Schipper et al ldquoTriggered contentrelease from optimized stealth thermosensitive liposomesusing mild hyperthermiardquo Journal of Controlled Releasevol 143 no 2 pp 274ndash279 2010

[19] M W Shahid A M Buchner M G Heckman et al ldquoDi-agnostic accuracy of probe-based confocal laser endomicro-scopy and narrow band imaging for small colorectal polyps afeasibility studyrdquo American Journal of Gastroenterologyvol 107 no 2 pp 231ndash239 2012

[20] L -iberville S Moreno-Swirc T Vercauteren E PeltierC Cave and G Bourg Heckly ldquoIn VivoImaging of thebronchial wall microstructure using fibered confocal fluo-rescence microscopyrdquo American Journal of Respiratory andCritical Care Medicine vol 175 no 1 pp 22ndash31 2007

[21] M de Smet S Langereis S v den Bosch and H GrullldquoTemperature-sensitive liposomes for doxorubicin deliveryunder MRI guidancerdquo Journal of Controlled Release vol 143no 1 pp 120ndash127 2010

[22] A Wei J G Mehtala and A K Patri ldquoChallenges andopportunities in the advancement of nanomedicinesrdquo Journalof Controlled Release vol 164 no 2 pp 236ndash246 2012

[23] M Derieppe B D de Senneville H Kuijf C Moonen andC Bos ldquoTracking of cell nuclei for assessment of in vitrouptake kinetics in ultrasound-mediated drug delivery usingfibered confocal fluorescencemicroscopyrdquoMolecular Imagingand Biology vol 16 no 5 pp 642ndash651 2014


[24] A Buades B Coll and J M Morel ldquoA review of imagedenoising algorithms with a new onerdquo Multiscale Modelingand Simulation vol 4 no 2 pp 490ndash530 2005

[25] N Cressie Statistics for Spatial Data Wiley-Interscience1993

[26] M Derieppe A Yudina M Lepetit-Coiffe B D de SennevilleC Bos and C Moonen ldquoReal-time assessment of ultrasound-mediated drug delivery using fibered confocal fluorescencemicroscopyrdquo Molecular Imaging and Biology vol 15 no 1pp 3ndash11 2013

[27] P Philipson and P Schuster Modeling by Non-Linear Dif-ferential Equations Dissipative and Conservative ProcessesWorld Scientific Publishing Co Pte Ltd vol 69 2009

[28] I Pratikakis C Barillot P Hellier and E Memin ldquoRobustmultiscale deformable registration of 3d ultrasound imagesrdquoInternational Journal of Image and Graphics vol 03 no 04pp 547ndash565 2003

[29] T Corpetti E Memin and P Perez ldquoDense estimation offluid flowsrdquo IEEE Transactions on Pattern Analysis andMachine Intelligence vol 24 no 3 pp 365ndash380 2002

[30] G M -urber and R Weissleder ldquoA systems approach fortumor pharmacokineticsrdquo Plos One vol 6 no 9 pp 1ndash102011

[31] T L Jackson ldquoIntracellular accumulation and mechanism ofaction of doxorubicin in a spatio-temporal tumor modelrdquoJournal of eoretical Biology vol 220 no 2 pp 201ndash2132003

[32] E A Swabb J Wei and P M Gullino ldquoDiffusion andconvection in normal and neoplastic tissuesrdquo Cancer Re-search vol 34 no 10 pp 2814ndash22 1974

[33] L Li T L M ten Hagen M Bolkestein et al ldquoImprovedintratumoral nanoparticle extravasation and penetration bymild hyperthermiardquo Journal of Controlled Release vol 167no 2 pp 130ndash137 2013

[34] A M Ponce B L Viglianti D Yu et al ldquoMagnetic resonanceimaging of temperature-sensitive liposome release drug dosepainting and antitumor effectsrdquo JNCI Journal of the NationalCancer Institute vol 99 no 1 pp 53ndash63 2007

[35] A I Minchinton and I F Tannock ldquoDrug penetration in solidtumoursrdquo Nature Reviews Cancer vol 6 no 8 pp 583ndash5922006

[36] H T Nia H Liu G Seano et al ldquoSolid stress and elasticenergy as measures of tumour mechanopathologyrdquo NatureBiomedical Engineering vol 1 no 4 pp 1ndash11 2016

[37] C D Arvanitis V Askoxylakis Y Guo et al ldquoMechanisms ofenhanced drug delivery in brain metastases with focusedultrasound-induced blood-tumor barrier disruptionrdquo Pro-ceedings of the National Academy of Sciences vol 115 no 37pp E8717ndashE8726 2018


Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

allowed the design of new drug delivery strategies with a localtriggering by an external source of mild hyperthermia -esedrug delivery systems must fulfill four main criteria (1) a longplasma half-life (2) a stable formulation at body temperature(3) a phase transition temperature corresponding to aphysiologically mild and safe hyperthermia hence the nameof low temperature-sensitive liposomes (LTSLs) and (4) aldquofastrdquo drug release rate from the liposomes when more than50 of their payload was released within around half a minutewhen exposed to their phase transition temperature [11 12]Needham and coworkers were the first to incorporate lyso-lipids in themembrane bilayer and paved the way for an LTSLformulation that was clinically compatible [13]

Combination of LTSLs with an external heat sourceallowed local treatment and Manzoor et al introduced theconcept of triggered rapid intravascular release [14] uponexternal triggering the LTSLs release the drug payloadrapidly in the tumor vasculature and build up a drugconcentration gradient between the tumor vasculature andthe tumor tissue which constitutes the driving force ofdiffusion that is the main source of transport for smallmolecules eg DOX [15] -is drug penetration is pro-portional to the drug concentration gradient Using TSLswith a fast drug release rate one can therefore maximizedrug penetration into the tumor

To conduct drug distribution studies at the tissue scaledorsal skin-fold window chambers have been used in vivo-ese consist of a skin flap held vertically with an aluminiumsaddle that is sutured to the upper and the lower parts of theflap [16ndash18] An optical window gives access to the fascialtissue and its vasculature and tumor cells can be implantedinto this window chamber An alternative intravital imagingsolution is fibered confocal fluorescence microscopy(FCFM) this modality allows real-time fluorescence imag-ing in a minimally invasive fashion by directly placing afiber-based optical probe in contact with the tissue of interestin its natural physiological environment FCFM is mainlyused in clinical applications involving hollow organs like ingastroenterology [19] and pulmonology [20] either visu-alizing the tissue based on its autofluorescence or afterinjection of fluorescein or indocyanine green

In this study we tested the feasibility to use FCFM tomonitor in vivo in real-time DOX penetration in the tumorinterstitium after intravascular release of DOX from the TSL(-ermodoxreg) -e kinetic analysis from the time seriesallowed quantifying (1) the local uptake kinetics of releasedDOX in each individual cell of the interstitium after releasefrom the TSL (2) the kinetics of the apparent released-DOXpenetration using the transport equation and (3) thereleased-DOX deposition the vascular washout and thedrug diffusion by means of an evolution model from thefluorescence signal intensity

2 Materials and Methods

21 Experimental Setup

211 Animals and Tumor Model All procedures wereperformed according to the ethical guidelines and were

approved by the animal welfare committee of UtrechtUniversity (DEC 2014III03035 Utrecht the Netherlands)WAGRij rats were purchased from Charles River (CologneGermany) -ey were maintained at room temperature with12 h light cycle in individually ventilated isolation cages andwere fed ad libitum -e rats were 12weeks old at the be-ginning of the experiments weighing 250 g Under gaseousanesthesia (Aerrane Baxter Deerfield IL) a skin incision ofa few millimeters was performed at the hind leg Sub-sequently rat R1 rhabdomyosarcoma tumor pieces (1ndash3mm3) were subcutaneously implanted in the hind leg usinga trocar When the tumor volume reached 1500 microm3 ap-proximately after 3weeks of tumor growth the drug ad-ministration and imaging experiments were performed

212 Chemicals Lysothermosensitive liposomal formula-tion of DOX (-ermodoxreg-TSL) at 2mgmL was obtainedfrom Celsion Corp (Lawrenceville NJ USA) -ese nano-particles release their payload as a burst in the temperaturerange of 395degC to 42degC ie less than 5 of release at 37degCand more than 65 at 41degC within about 30 sec (in vitro dataprovided by Celsion Corp) On the day of the real-timemonitoring experiment the -ermodoxreg solution was fil-tered using a PD10-desalting column (GE HealthcareEurope GmbH Eindhoven the Netherlands) to ensure thatthe DOX penetration that was monitored was fully encap-sulated previously in the TSL -e rodents were adminis-tered intravenously with a -ermodoxreg dose of 4mgkg

Doxorubicin hydrochloride (Sigma-Aldrich St-LouisMO) (relative molecular mass 580Da) named ldquofreeDOXrdquo in this study was injected intravenously at 4mgkg

An intravascular fluorescence label AngioSense 680 EXwas purchased from Perkin Elmer (Waltham MA USA)AngioSense is a 70 kDa near-infrared labeled-fluorescentpolymer (excitationemission wavelengths 670690 nm)which allows imaging the blood pool during the wholeimaging session

213 Fibered Confocal Fluorescence MicroscopyFluorescence images were acquired in real-time (85Hz) for20minutes using a dual-band FCFM system (Cellvizioregdual-band Mauna Kea Technologies Paris France) Nativefluorescence of DOX was collected with the 488 nm exci-tation channel henceforth referred to as ldquogreen channelrdquoand blood vessels via AngioSense with the 660 nm channelreferred to as ldquored channelrdquo -eir spectral sensitivity is500ndash630 nm and 680ndash800 nm respectively A 15 mm di-ameter FCFM microprobe (PF-2210 Mauna Kea Technol-ogies) was used (Figure 1(c)) with the following imagingspecifications lateral resolution 33 μm field of view (FOV)602 μmtimes 602 μm axial resolution 15 μm working distance0 μm-e tissue was excited at 4mW of laser power for bothchannels

214 Animal Positioning A water bath (MemmertSchwabach Germany) was used to ensure a mild and ho-mogeneous tumor heating (Figure 1(b)) -e water









(a) (b)

5 mm

(c)

Anesthesia

10 s 50 s











(d)















1113960 1113961 (1)





It + Vrarr

middot nablararr

I 0 (2)









argminVrarr

1113946Ω

1113868111386811138681113868It + Vrarr

middot nablararr

I1113868111386811138681113868 + α1113874

nablararr

u2

2 +nablararr

v2

21113875 d rrarr

(3)





u2

2 u2x + u2

y andnablararr

v2

2 v 2x + v 2

y ux uy vx with vy







z

ztS( r


rarr) middot S( r


rarr t) (4)






t)





5 Results












6 Discussion







0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)



0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)



Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























(a) (b)

5 mm

(c)

Anesthesia

10 s 50 s











(d)















1113960 1113961 (1)





It + Vrarr

middot nablararr

I 0 (2)









argminVrarr

1113946Ω

1113868111386811138681113868It + Vrarr

middot nablararr

I1113868111386811138681113868 + α1113874

nablararr

u2

2 +nablararr

v2

21113875 d rrarr

(3)





u2

2 u2x + u2

y andnablararr

v2

2 v 2x + v 2

y ux uy vx with vy







z

ztS( r


rarr) middot S( r


rarr t) (4)






t)





5 Results












6 Discussion







0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)



0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)



Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































1113960 1113961 (1)





It + Vrarr

middot nablararr

I 0 (2)









argminVrarr

1113946Ω

1113868111386811138681113868It + Vrarr

middot nablararr

I1113868111386811138681113868 + α1113874

nablararr

u2

2 +nablararr

v2

21113875 d rrarr

(3)





u2

2 u2x + u2

y andnablararr

v2

2 v 2x + v 2

y ux uy vx with vy







z

ztS( r


rarr) middot S( r


rarr t) (4)






t)





5 Results












6 Discussion







0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)



0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)



Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















u2

2 u2x + u2

y andnablararr

v2

2 v 2x + v 2

y ux uy vx with vy







z

ztS( r


rarr) middot S( r


rarr t) (4)






t)





5 Results












6 Discussion







0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)



0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)



Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























6 Discussion







0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)



0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)



Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















0 min

50 μm

(a) 0 min (b)

4 min(c) 4 min (d)

8 min(e) 8 min (f)



0 40 80 120 160 2000 30 60 90 120 150(au)

50 μm

(i)



Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Le Right

0

2

4

6

8

10

12

Upt

ake r

ate 1

k (m

in)

p lt 0001


(a)

(d)

(b)

(e)

(g)

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 1

2 4 6 8 10 12 14 16 18 20

50

100

150

200


Fluo

resc

ence

sign

al in

tens

ity (a

u)


Nucleus 2

(c)

(f)

(au)50

100

150

200


min0

1

2

3

4

5

6

7

8

910

Le Right

Field of view

0

50

100

150

200

250

300

Max

imum

DO

X flu

ores

cenc

e int

ensit

y (a

u)

Field of view

2

4

6

8

10

12

14

Upt

ake r

ate 1

k (m

in)

2







Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























Disp

lace

men

t fie

lds

DO

X flu

ores

cenc


0

50

100

150

(au)

Eigenvector 2(6)

Eigenvector 1(94)

(k)

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)


0 5 10 15 20




minus10

minus8

minus6

minus4

minus2

0

2Av

erag

ed am

plitu

de o

f disp

lace

men

t (μm

)

(l)



0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















0

50

100

150

Mea

sure

d da

ta

(a) (b) (c) (d)


1000 2000 3000 4000 5000

0052

0053

0054

0055

0056


Resid

ue o

f the

leas

t-squ

are f

it (a


(m)



Dru

g de

posit

ion

(au

)

14

16

18

2

22

(n)



Vasc

ular

was

hout

(au

)

016

018

02

022

024

026

028

(o)

Mod

eled

dat

a

(e) (f) (g) (h) 0

50

100

150

Resid

uals

(i) (j) (k) (l)(au)

0

50

100

150










7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























7 Conclusions


Data Availability




Acknowledgments





References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Assessment of Intratumoral Doxorubicin Penetration after ...described in Derieppe et al. [23]. Brieﬂy, this automated pipeline includes cell detection, which was facilitated by a