A Targeting Microbubble for Ultrasound Molecular Imaging
A Targeting Microbubble for Ultrasound Molecular Imaging
James Shue-Min Yeh 0 1
Charles A. Sennoga 0 1
Ellen McConnell 0 1
Robert Eckersley 0 1
Meng-Xing Tang 0 1
Sussan Nourshargh 0 1
John M. Seddon 0 1
Dorian O. Haskard 0 1
Petros Nihoyannopoulos 0 1
0 1 National Heart and Lung Institute, Imperial College London , London , United Kingdom , 2 Department of Cardiology, Hammersmith Hospital , London , United Kingdom , 3 Imaging Sciences Department, Medical Research Council, Imperial College London , London , United Kingdom , 4 Department of Chemistry, Imperial College London , London , United Kingdom , 5 Department of Bioengineering, Imperial College London , London , United Kingdom , 6 William Harvey Research Institute, Queen Mary, University of London , London , United Kingdom
1 Editor: Christoph E Hagemeyer, Baker IDI Heart and Diabetes Institute , AUSTRALIA
Microbubbles conjugated with targeting ligands are used as contrast agents for ultrasound
molecular imaging. However, they often contain immunogenic (strept)avidin, which
impedes application in humans. Although targeting bubbles not employing the
biotin(strept)avidin conjugation chemistry have been explored, only a few reached the stage of
ultrasound imaging in vivo, none were reported/evaluated to show all three of the following
properties desired for clinical applications: (i) low degree of non-specific bubble retention in
more than one non-reticuloendothelial tissue; (ii) effective for real-time imaging; and (iii)
effective for acoustic quantification of molecular targets to a high degree of quantification.
Furthermore, disclosures of the compositions and methodologies enabling reproduction of
the bubbles are often withheld.
To develop and evaluate a targeting microbubble based on maleimide-thiol conjugation
chemistry for ultrasound molecular imaging.
Microbubbles with a previously unreported generic (non-targeting components)
composition were grafted with anti-E-selectin F(ab’)2 using maleimide-thiol conjugation, to produce
E-selectin targeting microbubbles. The resulting targeting bubbles showed high specificity
to E-selectin in vitro and in vivo. Non-specific bubble retention was minimal in at least three
non-reticuloendothelial tissues with inflammation (mouse heart, kidneys, cremaster). The
bubbles were effective for real-time ultrasound imaging of E-selectin expression in the
inflamed mouse heart and kidneys, using a clinical ultrasound scanner. The acoustic signal
intensity of the targeted bubbles retained in the heart correlated strongly with the level of
Competing Interests: The authors have read the
journal's policy and have the following competing
interests: JY was recipient of the Bristol-Myers
Squibb Cardiovascular Prize Fellowship. JY is
applicant of patents pending on Microparticle
Compositions (UK Patent Application GB1223332.6
and GB1300064.1; International Patent Application
reference PCT/GB2013/053398). These do not alter
the authors' adherence to PLOS ONE policies on
sharing data and materials.
Targeting microbubbles for ultrasound molecular imaging, based on maleimide-thiol
conjugation chemistry and the generic composition described, may possess properties (i)–(iii)
desired for clinical applications.
Ultrasound molecular imaging has been achieved using targeting microbubbles which contain
targeting ligands on the bubble-shell . Following intravenous (iv) administration, these
echogenic bubbles circulate and accumulate in regions expressing the molecules targeted,
depicted on ultrasound pictures as areas of bright signals locating the molecules of interest.
This technique has allowed molecular imaging of pathophysiological processes such as
inflammation, angiogenesis and thrombosis, and has potential for clinical applications (Table A in
However, targeting bubbles often employed biotin-(strept)avidin conjugation chemistry
for grafting targeting ligands to the bubble-shell (Table A in S1 File), which is best avoided
for use in humans because of the high immunogenicity of (strept)avidin. Targeting bubbles
not employing the biotin-(strept)avidin conjugation chemistry have been developed, these
included those based on maleimide-thiol or other conjugation chemistries (reviewed in Yeh
2010) . Only about half of them were tested in animals for ultrasound molecular imaging
(Table A in S1 File), none were reported/evaluated to show all three of the following properties
desired for clinical applications: (i) low degree of non-specific bubble retention in more than
one non-reticuloendothelial tissue (allowing specific molecular detection in different tissues);
(ii) effective for real-time imaging (allowing ready visualization of the molecular target(s) by
the bedside, and better assessment of moving objects such as the beating heart); and (iii)
effective for quantification of molecular targets to a high degree of quantification (increasing
diagnostic power). Furthermore, enabling disclosure of the compositions and methodologies for
reproducing these bubbles are often withheld (Table A in S1 File).
In this study, we developed and disclosed the full methodology for producing a targeting
microbubble, based on maleimide-thiol conjugation chemistry and a previously unreported
generic (non-targeting components) composition. E-selectin (Esel), an adhesion molecule
expressed on the endothelium in inflammation, was chosen as a prototype molecule for bubble
targeting. We showed that the Esel targeting bubble developed was effective for molecular
imaging in inflammation of the heart and kidneys in vivo. The bubble possessed all three
properties (i)-(iii). Based on the maleimide-thiol conjugation chemistry and properties (i)-(iii), the
bubble is a potentially candidate for further development towards clinical translation.
MES-1 monoclonal antibody (mAb), a rat IgG2a,κ against mouse Esel, and its F(ab’)2 fragment
were provided by Dr D Brown (UCB-Celltech, UK) . MES-1 labelled with 7 Alexa Fluor 488
fluorescence dye (AF488-MES-1) and reduced MES-1 F(ab’)2 containing 2 thiol groups per F
(ab’)2 from tris(2-carboxyethyl)phosphine hydrochloride reduction was prepared as described
in the S1 File. MEC13.3 mAb, a rat IgG2a,κ against mouse PECAM-1 (BD Biosciences),
allophycocyanin-labelled mAb against mouse PECAM-1 (BD Pharmingen), rat IgG2a,κ isotype
negative control mAb (BD Biosciences) and biotinylated rabbit mAb against rat IgG2a (Vector
Laboratories) were purchased.
Wild-type (WT) mice were adult male C57BL/6J (Charles River, UK). Esel knock-out (KO)
mice were adult male Esel homozygote KO on C57BL/6J background , bred locally from
mice donated by Dr K Norman and Prof P Hellewell (University of Sheffield, UK). All animal
work was carried out under licences granted by the UK Home Office under the Animals
(Scientific Procedures) Act 1986. All animals were sacrificed humanely by dislocation of the neck, or
overdose of anesthetic followed by dislocation of the neck. Ethical approval was obtained from
Imperial College London's Ethical Review Panel.
Mouse Model of Lipopolysaccharide-Induced Systemic Inflammation
WT and Esel KO mice were treated with 50μg lipopolysaccharide (LPS) from E Coli 0111:B4
(Sigma-Aldrich), made up to 200μL volume in normal saline, by intraperitoneal (ip) injection
to induce systemic inflammation .
Immunohistochemistry was performed on acetone-fixed cryosections of freshly harvested
hearts of the WT (with/without LPS pre-treatment) and Esel KO (LPS pre-treated) mice, using
a standard protocol detailed in the S1 File. The primary antibodies used were MES-1 (for Esel),
MEC13.3 (for PECAM-1, an endothelial marker) and rat IgG2a,κ isotype negative control
mAb. Color was developed using 3,30-diaminobenzidine with hematoxylin counterstaining.
Reverse Transcriptase—Real Time Quantitative Polymerase Chain
WT mice were pre-treated with LPS as described above. The duration between LPS treatment
and animal sacrifice for immediate tissue harvesting was noted as the LPSTime. RNA extraction
and RT-qPCR for Esel and hypoxanthine phosphoribosyltransferase-I (HPRT-I, a house
keeping gene) in the hearts were performed; only apical-half of the harvested hearts were used in
order to avoid measuring Esel from the great vessels. Note that Esel is expressed on endothelial
cells and not on other cell types. RT-qPCR was performed using standard protocols and kits
according to the manufacturers’ instructions. All PCR reactions were carried out in triplicates
on a 96-well plate. Means of the replicates were used. Esel mRNA concentration was expressed
as % HPRT-I. Further details in S1 File.
A generic (non-conjugated) microbubble was first produced by sonicating C3F8-sparged
aqueous suspension containing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti
Polar Lipids, AL),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide(polyethylene glycol)-2000) (DSPE-PEG2000-Maleimide; Avanti Polar Lipids), mono-stearate
poly(ethylene)glycol (PEG40-stearate; Sigma-Aldrich), and fluorescent dye
1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) at 75:9:14:2 molar ratio.
Reduced MES-1 F(ab’)2 containing 2 thiol groups per F(ab’)2 (prepared as described in S1 File)
were then linked to maleimides on the bubble shell outer-surface by maleimide-thiol
conjugation to produce the Esel targeting bubble. The conjugation reaction ratio was 4.338x106 F(ab’)2
molecules per bubble, estimated F(ab’)2:maleimide reaction molar ratio 10:1 (see S1 File).
The reaction was terminated by adding excess N-Ethylmaleimide (NEM, Sigma-Aldrich) to
quench any unreacted thiols. Bubbles before and after conjugation were washed with cold
degassed normal saline several times, by centrifugation-flotation with exchange of the
subnatents, to remove unincorporated components (including excess F(ab’)2 and NEM) and bubble
fragments. In previous experiments, we found that by keeping the conjugation sites on each
ligand to a minimum (ie, reducing only 1 inter-chain disulfide bond to produce 2 thiols per F
(ab’)2), and inactivating any unreacted thiols after bubble conjugation (eg, alkylation of
unreacted thiols using NEM), significant bubble aggregation due to cross-linking was avoided
. Further details in S1 File.
Bubbles were diluted (eg, 1:200) in cold normal saline and examined under microscopy in a
hemocytometer. Bubble concentration and size distribution were determined by electrozone
sensing in a Coulter Multisizer IIe equipped with a 30μm-diameter orifice counting tube
(Coulter Electronics), according to the manufacturer’s instructions. The electrozone sensing has
detection range 0.72–18μm, resolution 0.09μm. All bubble concentrations and calculated
dosages in this study were based on electrozone sensing (nb, different methods such as
microscopy, electrozone sensing or laser diffraction, give different particle concentration and size
distribution , due to differences in the nature of the analytical process and the minimum
size detectable). For bubble charge analysis, the bubbles were dispersed in 1mL of 1mmol/L
KCl (pH 7.4) at 107 bubbles/mL as described by others [7, 8]. Bubble net charge was
determined as the zeta potential by light scattering in a Zetasizer Nano ZS (Malvern Instruments),
according to the manufacturer’s instructions.
Targeting Microbubble Binding Assay In Vitro
100μL of Esel targeting or non-targeting (generic) bubbles at 2.5x107 bubbles/mL were placed on
inverted polystyrene petri-dishes coated with 200μL of recombinant homodimeric mouse Esel
protein (R&D Systems) at 7nmol/L (dish E), or on Esel coated dishes previously blocked with
500μL of excess MES-1 F(ab’)2 at 67nmol/L (dish B), or on non-coated dishes where phosphate
buffered saline pH 7.5 (PBS) was used instead of Esel for dish coating (dish P). Unattached
bubbles were gently washed off after 1min. The dishes were then re-filled with cold degassed PBS for
immediate examination under an upright light microscope equipped with immersion objective
lens. The number of bubbles attached on each dish was counted and averaged from 10 random
optical fields (OFs) to determine the attached bubble density. Further details in S1 File. Different
batches of bubbles (made at different times) were tested on different occasions. To correct for
experimental variations amongst the different occasions, the attached bubble density was
normalised against that of the targeting bubbles on dish E in the corresponding occasion.
WT and Esel KO mice were pre-treated with 50ng recombinant IL-1β (R&D Systems)
intrascrotally to induce cremaster inflammation, 2 hours (h) before general anesthesia (xylazine/
ketamine mixture ip) and exteriorization of the muscle for intravital microscopy. 1.5x107 Esel
targeting bubbles in 100μL normal saline were administered as a rapid iv bolus through the tail
vein catheter of these animals, followed by a 100μL normal saline flush, for intravital
microscopy in the cremaster. Observations were made using an upright microscope equipped for
bright-field and fluorescence microscopy, with 20x and 40x immersion objective lens,
chargecoupled device (CCD) and silicon intensifier target (SIT) cameras. See S1 File for detailed
setup. Blood flow and bubbles were assessed over several OFs encompassing a number of different
vessels in different vascular beds (arteries, veins, capillaries) under bright-field and
fluorescence microscopy. The number of freely circulating bubbles in a monitor OF were counted
over 10s under fluorescence microscopy at 5, 7, 10 ± 15min after bubble injection. The
accumulation of attached bubbles (defined as not moving for >3s) in an OF field were assessed for
up to 15min post bubble injection. At 15min (when freely circulating bubbles were absent/
minimal), multiple OFs were used to assess the number of attached bubbles in 20–40μm
diameter venules: one to five 400μm-length segments of 2–6 venules were examined per animal. In
some animals, the attached bubbles in the same OF were assessed for up to 90min under
intermittent combined bright-field and fluorescence microscopy, looking for cellular internalization
or transmigration into the tissue interstitium. Shear rates against bubble attachment were
determined from microvascular center-line red blood cell velocities (Vrbc) in 5 random
segments of 20–40μm diameter venules, using an Optical Doppler Velocimeter (Microcirculation
Research Institute, Texas A&M University, Texas), before bubble injection. All animals were
sacrificed at the end of the experiment. For analysis, the density of attached bubbles was
expressed as the number of bubbles per vessel surface area (VSA), where VSA = πDL, D and L
are the vessel segment diameter and length, respectively. The attached bubble density for each
venule was taken as the mean of its segments, and that for each animal was taken as the mean
of its venules. Shear rate was calculated using: Shear rate ¼ 8 VDb, where Vb ¼ Vrabc, Vb is the mean
bulk velocity, α is the factor converting Vrbc to Vb (taken as 1.6 for Poiseuille flow in veins).
The shear rate for each animal was taken as the mean of the 5 random venule-segments
1.5x107 Esel targeting bubbles were administered to WT and Esel KO mice pre-treated 2.5h
before with 50ng IL-1β intrascrotally, as described in intravital microscopy. 15min post bubble
injection, a rapid iv bolus of a 150μL cocktail containing 50μg AF488-MES-1 (against Esel)
+ 25μg allophycocyanin-labelled mAb (against PECAM-1, an endothelial marker) in normal
saline was administered, followed by a 100μL normal saline flush. After a further 15–20min,
animals were given terminal anesthesia by xylazine/ketamine mixture iv, followed by manual
perfusion with PBS to remove unattached bubbles and mAb from the circulation. Immediately
thereafter, the cremasters were harvested and fixed in 4% paraformaldehyde PBS for confocal
microscopy. 3 different fluorescence were scanned: DiI for bubbles, Alexa Fluor 488 for Esel
and allophycocyanin for PECAM-1. Further details in S1 File.
15 WT and 8 Esel KO mice were imaged. All were pre-treated with LPS. Ultrasound imaging
was performed under ip xylazine/ketamine general anesthesia. The Acuson Sequoia 512 clinical
ultrasound scanner equipped with a 15L8-s linear array transducer (Siemens, CA) was used.
Gel was coupled between the shaven skin and the transducer. 14MHz contrast pulse
sequencing (CPS) mode imaging at low power (mechanical index (MI) = 0.22–0.26), dynamic range
55dB was used. Gain and other settings were fixed. Bubble signals were presented in heated
object scale (‘CPS-contrast only’ images), tissue signals in grey scale (‘B-mode’ images).
Baseline images of the heart in the parasternal short axis (PSA) papillary muscle level, parasternal
long axis (PLA) and apical 4-chamber (A4C) views were acquired before bubble
administration. Imaging was then maintained in the PSA view by fixing the transducer in position with a
free standing clamp. A stopwatch was started and 108 Esel targeting bubbles (in 100μL volume
made up with normal saline) injected at 10s as an iv bolus over 1–2s through a cannula in the
tail vein. This was followed by a 100μL normal saline flush at 20s. To capture real-time sequence
of events and detect Esel expression in the heart, continuous ultrasound imaging was performed
and recorded as 3s-digital clips, starting at time 0 on the stopwatch and repeated at
pre-determined time intervals. To determine the nature of bubble signal attenuation, the following was
performed in addition: (i) other views of the heart (PLA, A4C) were acquired at the end; (ii)
7MHz CPS imaging at MI 0.22 (keeping the gain & other settings the same as 14MHz imaging)
was acquired at baseline & end of the 14MHz imaging in some animals; and (iii) wider PSA
view to include other structures surrounding the heart was acquired at 5min intervals. To
image extra-cardiac tissues, 14 and 7MHz CPS imaging of the thorax, abdomen and pelvis were
also performed in the antero-posterior plane at baseline & end of the cardiac imaging study in
some animals. To do this, the probe was positioned transversely and moved slowly caudal from
just below the neck to the pelvis during image recording. All animals received only one dose of
bubbles to negate carry-over effects from previous bubble dosing (eg, blocking of Esel binding
sites). The duration between LPS treatment and the administration of bubbles was noted as the
LPSTime. All animals were sacrificed at the end. Further details in S1 File.
Acoustic Quantification of Esel Expression in the Heart
As circulating bubble signal in the left ventricular (LV) cavity (blood pool) was absent/minimal
by 20min post bubble administration in all animals, the bubble signal intensity in the
myocardium at 24min was assessed for acoustic quantification of Esel expression in the heart. To
do this, several end-diastolic image frames of the heart (‘CPS-contrast only’ images) within the
3s-recording period at 24min 10s post bubble administration were selected and aligned. A
videodensitometric method was used to quantify the CPS bubble signal intensities in the
anterior wall of the myocardium (region of interest M), using the YABKO software (Charlottesville,
Virginia). This region was chosen because it was consistently least/minimally affected by
ultrasound attenuation in all animals. The CPS video signal intensities (VI’s) were ‘linearized’ by
log-decompression using the formula: Linearized VI ¼ 255 10ðVI252555 Dynam2i0c RangeÞ. The linearized
VI (I) was expressed in arbitrary acoustic units (AU). I’s of the images were averaged, then
subtracted by average I of the baseline (before bubble administration) images. The
baseline-subtracted I in the myocardium at 24min 10s post bubble administration (R24) represented the
retained bubble signal intensity in the myocardium at 24min 10s post bubble administration.
As the circulating bubble signal intensities in the myocardium at 24min 10s were negligible (ie,
a circulating bubble signal intensity of <0.1 AU in the LV cavity (blood pool) at this time
would contribute an undetectable circulating bubble signal intensity of <0.005–0.024 AU in
the myocardium, due to a relative myocardial blood volume of 5–24%) [9–11], their
subtraction from R24 was not required. For data analysis, R24 was correlated against the level of Esel
expression in the heart, in terms of LPSTime or Esel mRNA concentration by qRT-PCR. Note:
(i) The Esel mRNA concentration was determined from a standard curve of LPSTime vs Esel
mRNA concentration in the hearts of 42 mice (LPSTime range: 3–16h) . Due to the relatively
long duration of each imaging study (mean 45–60min from the time of bubble
administration to tissue harvesting), it was not possible to correlate R24 of individual animals against its
own Esel expression level determined ex-vivo by independent means (eg, RT-qPCR), because
Esel expression decreased rapidly with time in the mouse model used [2, 5], such that the
retained bubble signal intensities reflected Esel expressions around the time of bubble
administration rather than tissue harvest. (ii) We have shown previously that the relationships between
the cell-surface Esel protein (actual bubble target) concentration and LPSTime (for LPSTime
3h) or Esel mRNA concentration in the heart were curvilinear; and approximately linear in
the range of LPSTime’s (4–6h) or mRNA concentrations (50–220% HPRT-I) imaged in this
study [2, 5]. (iii) As Esel expression in the myocardium was essentially global and uniform in
the mouse model used , the quantification of Esel expression from the anterior myocardial
wall by ultrasound, and from the apical-half of the heart by qRT-PCR, were regarded as
equivalent (both represented that of the whole heart).
Pearson correlation was performed, where indicated. Student’s t-test or one-way ANOVA with
Tukey’s post-hoc analysis was used for significance testing, where appropriate, with p<0.05
taken as statistically significant.
Esel Targeting Microbubbles
A schema of the Esel targeting bubbles is shown in Fig 1. The bubbles showed spherical
morphology; bubble-bubble aggregation or cross-linking was minimal as assessed qualitatively on
microscopy, Fig 2A. The bubble size distribution was reproducible amongst 5 batches prepared
on separate occasions; the mean (SEM) bubble diameter was 2.2 (0.2) μm, 98.6% or 100% of
the bubble population were under 6 or 10μm in diameter, respectively, Fig 2B. The bubbles had
a near neutral charge, zeta potential = 5mV at pH 7.4.
In Vitro Binding of Esel Targeting Microbubbles
Esel targeting bubbles attached to Esel coated on dish (dish E): the mean (SEM) attached
bubble density was 2060 (1070) bubbles/mm2 for the 5 batches of targeting bubbles produced on
separate occasions, Fig 2C. The specificity of the targeting bubbles to Esel was demonstrated by
the abolishment of bubble attachment when Esel was pre-blocked with excess anti-Esel
antibody (F(ab’)2) (dish B). Additional negative controls using targeting or non-targeting bubbles
(generic non-conjugated bubbles) on dishes not coated with Esel (dish P), or non-targeting
bubbles on dish E and B showed similar low levels of bubble attachment. One-way ANOVA
with Tukey’s post-hoc analysis showed significant differences between targeting bubbles on
dish E and the negative controls (p<0.0001); no significant difference was observed amongst
the negative controls.
In Vivo Validation of Esel Targeting Microbubbles
Esel targeting bubbles were administered to 5 WT (body weight mean (SD, range): 25 (2, 23–
27) g) and 5 Esel KO (24 (2, 22–27) g) mice at 3.1–4.1h and 3.3–4.5h post IL-1β treatment,
respectively. Under intravital microscopy, the bubbles were seen to circulate and reach the
cremaster muscle 7–17s post bubble administration. The bubbles attached and accumulated in
the cremaster venules of the WT mice (mean (SEM) attached bubble density = 370 (46)
bubbles/mm2 VSA), this was minimal in the KOs (11 (3) bubbles/mm2 VSA), p<0.0001 (student’s
t-test), suggesting high targeting specificity and low degree of non-specific bubble attachment/
retention, Fig 3A and 3B (S1 and S2 Videos). Bubble attachment in the arteries, arterioles,
Fig 1. Schema of Esel targeting microbubble.
capillaries or large veins was absent/minimal. Rolling was observed in a small minority of
bubbles; complete detachment of the attached bubbles was infrequently seen. Intravascular
obstruction by the bubbles and bubble attachment to leukocytes were not detected.
Transmigration into the tissue interstitium or cellular internalization of the attached bubbles was also
not detected, when observed for up to 90min.
Confocal microscopy showed co-localization of the targeted bubbles with endothelial
cellsurface Esel in the WT cremaster venules (n = 3 animals, body weight 21.8–24g), further
confirming Esel specificity of the targeting bubbles, Fig 3C. Esel expression was not detected in the
KOs (n = 2, body weight 25.7–28.4g).
The shear rates against bubble attachment were not significantly different between the WT
(mean (SEM) = 329 (46) s-1, n = 3) and KO (211 (37) s-1, n = 3) mice, p = 0.30 (student’s
ttest). The diameter of the vessels sampled for shear rates were similar between the two groups:
mean (SEM) vessel diameter 30 (3) μm in the WT vs 34 (1) μm in KO group, p = 0.12 (student’s
t-test). The mean body weight (SD, range) was 26 (2, 25–28) g and 27 (1, 26–29) g in the WT
and KO group, respectively.
Fig 4A showed that the number of freely circulating bubbles decreased exponentially with
time (the exponential nature supported first-order kinetics  in the elimination of the
bubbles). Circulating bubbles cleared from the blood pool sooner in the WTs than KOs,
presumably due to there being less circulating bubbles in the WTs, because a larger proportion of the
administered bubbles ended up as non-circulating bubbles due to their attachment to Esel in
the WTs. Circulating bubbles that do not become attached are eliminated by the
reticuloendothelial tissues (eg, liver, spleen) and lungs. In all animals, the circulating bubbles were minimal/
undetectable beyond 10min post bubble administration. The density of the attached bubbles
on the cremaster venules increased with time, reaching a plateau soon after ( 2min) iv bolus
administration of the bubbles, Fig 4B. In one WT animal (WT 3), excessive and prolonged
Fig 2. In vitro validation of Esel targeting microbubbles. (A) Bright-field microscopy of Esel targeting bubbles. (B) Size distribution of Esel targeting
bubbles. Plotted are the mean and SEM (error bar) for 5 batches of bubbles prepared on separate occasions. (C) In vitro binding of Esel targeting bubbles.
Plotted are the mean and SEM (error bar) of attached bubble densities relative to that of Esel targeting bubbles on dish E, n = 5 batches of Esel targeting
bubbles and 2 batches of non-targeting bubbles.
Fig 3. In vivo validation of Esel targeting microbubbles. Intravital microscopy of Esel targeting bubbles in the mouse cremaster. (A) Fluorescence
microscopy (SIT camera images, magnification 200x) of attached bubbles in a representative WT (i) and Esel KO (ii) mouse of Fig 3B (S1 and S2 Videos). (B)
Quantification of bubbles attached to the cremaster venule. Each point represents one animal; group mean and SEM (error bars) are shown; n = 5 WT and 5
Esel KO. (C) Confocal microscopy of Esel targeting bubbles in the mouse cremaster venule. Esel in green (i, ii), bubbles in red (iii, iv), endothelium
(PECAM1) in grey (v, vi), all 3 components combined (vii, viii).
Fig 4. In vivo kinetics of Esel targeting microbubbles. Intravital microscopy of Esel targeting bubbles in the mouse cremaster. (A) Elimination kinetics of
circulating bubbles. Group mean and SD (error bars) are plotted. (B) Accumulation kinetics of attached bubbles. N = 5 WT and 5 KO in both (A) & (B); these
are the same animals as those in Fig 3A and 3B.
light exposure in the OF was inadvertently applied >7 min post bubble administration—this
reduced subsequent fluorescence detection of the bubbles retained in that OF by the SIT
camera. As a result, bubble counts >7 min post bubble administration in that OF were unreliable
and therefore excluded from analysis in this animal.
1. Animals. 15 WT and 8 Esel KO mice were imaged. 3 WT mice were excluded from analysis
because of bubble dosing error (one animal) or uncertainties regarding the level of Esel
expression (two animals). Thus, 12 WT (mean body weight (SD, range) = 19.7 (1.4, 18–22)
g) and 8 KO (22.3 (3.7, 17–28) g) mice were analysed. The LPSTime ranged 3.9–6.0h in the
WT and 4.5–5.7h in the KO group.
2. Cardiac Imaging. Bright signal artefacts present both before and after bubble administra
tion could be seen in the WT and Esel KO animals; these artefacts were small and outside
the myocardium, Figs 5A and 6, and Fig A in S1 File.
The real-time sequence of events in the heart for a single iv bolus of the targeting bubbles is
shown in Fig 5a. Bubble signal was detected first in the right heart chambers within 4 heart
beats ( 1s) of bubble administration. This was followed by bubble signal appearing in the
left heart chambers as the bubbles returned from the pulmonary circulation. The bubble
signal intensities rose rapidly, peaking in the LV cavity and myocardium within 6–7 heart
beats ( 1.5–2s) and 9–12 heart beats ( 2–3s) after bubble administration, respectively. The
signal intensities then decreased over time.
Esel expression in the WT myocardium was visualized in real-time, best demonstrated by
the persistence of bubble signals (retention of attached bubbles) in the myocardium beyond
the clearance of circulating bubbles from the blood pool (LV cavity). In the KO
myocardium, the persistence of bubble signals was minimal, consistent with a minimal degree of
non-specific bubble retention, Figs 5A and 6.
Frozen section immunohistochemistry confirmed the presence and absence of Esel
expression in the LPS pre-treated WT and KO hearts, respectively. The spatial distribution of Esel
Fig 5. Real-Time Ultrasound Molecular Imaging of Esel Expression in the Mouse Heart. (A) Sequential 14MHz CPS images of the heart in end-diastole
PSA view from 00:10 to 24:20 post iv bolus administration of Esel targeting bubbles, in a (1) WT and (2) Esel KO mouse pre-treated with LPS, respectively.
Bubble signal is presented in a heated object scale. M: region of interest in the myocardium for acoustic quantification. A labelled diagram of the ultrasound
images is shown: LVC (left ventricular cavity), Myo (left ventricular myocardium), RVC (right ventricular cavity). (B) Frozen section immunohistochemistry for
Esel in the heart of a (1) WT and (2) Esel KO mouse. Positive staining = brown color. Controls for staining is shown in Fig A in S1 File. Magnification 200x.
expression was essentially uniform throughout the myocardium, but limited to the
capillaries and post-capillary venules, Fig 5B and Fig A in S1 File.
Significant ultrasound attenuations due to the high initial bubble concentrations occurred
early following bubble bolus administration, with major loss of signals in regions of the
heart located distally in the ultrasound path (eg, 5–10 o’clock positions in the myocardium
and adjacent LV cavity in the PSA view, Fig 5A). As the circulating bubble concentrations
in the blood pool decreased over time, the attenuations diminished (compare frame 0:30 vs
10:20 in Fig 5A) but did not disappear (frame 20:20 in Fig 5A1)—most likely due to
attenuations caused by overlying bone, lung air ± retained bubbles. This caused pseudo-loss of
targeted bubble signals for Esel in the WT mice. However, by changing the scan plane to alter
Fig 6. Real-Time Ultrasound Molecular Imaging of Esel Expression in the Mouse Heart. PSA, PLA and A4C views of the heart at 14 and 7MHz CPS,
>20min post bubble administration (when freely circulating bubbles have cleared from the blood pool (LV cavity)). Animal, gain and MI were the same
between both frequencies. Bubble signal is presented in a heated object scale. Arrow indicates recovery of retained bubble signal in the mid anteroseptal
wall by changing the scan plane from PSA to PLA or ultrasound frequency from 14 to 7MHz. Baseline images before bubble administration are shown in Fig
B in S1 File.
the relative positions of the overlying entities, or by lowering the ultrasound frequency to
increase its penetrative depth, these attenuations could be overcome with good ‘recovery’ of
the retained bubble signals, Fig 6. The global expression of Esel in the WT myocardium was
thus demonstrated on ultrasound imaging, consistent with the immunohistochemistry
3. Imaging of Other Tissues. Comparison of the thoracic, abdominal and pelvic scans
between the WT and Esel KO animals with reference to the baseline images (before bubble
administration), showed low/minimal non-specific retention of the targeting bubbles in
more than one non-reticuloendothelial tissues. Esel expression was detected in the renal
cortex of the WT but not KO animals (Fig 7)–the spatial distribution of the targeted signals
was consistent with the predominant expression of Esel in the glomeruli . As expected,
the targeting bubbles were taken up by the spleen and liver in both WT and KO animals, the
major reticuloendothelial tissues involved in bubble elimination.
4. Acoustic quantification of Esel Expression in the Heart. Acoustic quantification of the
level of Esel expression to a high degree of quantification was possible using R24, which
correlated strongly with the level of Esel expression in terms of LPSTime (r = -0.8) and Esel
mRNA concentration (r = 0.82) in the heart, Fig 8. The latter two previously shown to be
linearly related to the cell-surface Esel protein (actual bubble target) concentration in the
heart, in the expression range imaged .
Adverse Effects. No acute adverse effects were observed in all 38 animals following bubble
administration: intravital microscopy (5 WT, 5 KO), confocal microscopy (3 WT, 2 KO) and
ultrasound molecular imaging (15 WT, 8 KO).
In this early developmental study, a targeting bubble based on maleimide-thiol conjugation
chemistry with the generic (non-targeting components) composition was successfully
engineered. The bubble demonstrated all three of the following properties desired for clinical
applications: (i) low degree of non-specific retention in more than one non-reticuloendothelial
tissue; (ii) effective for ultrasound imaging in real-time; and (iii) effective for acoustic
quantification of the targeted molecule to a high degree of quantification. Based on the
maleimidethiol conjugation chemistry and properties (i)-(iii), the bubble is a potential candidate for
further development towards clinical translation.
The generic composition of our bubble consisted of C3F8-gas encapsulated within a
phospholipid-shell made from an aqueous suspension of DSPC:DSPE-PEG2000-Maleimide:
PEG40-stearate at 75:9:14 molar ratio (a small amount of DiI fluorescent dye was added for
bubble visualization under intravital/confocal microscopy). The same composition has not
been reported in other bubbles, although frequently such information is not available or
insufficiently disclosed. Where known, the composition differs to ours in the number, type,
combination, or molar ratio of the chemical components used (Table A in S1 File).
The maleimide-thiol conjugation chemistry has the potential advantage of low
immunogenicity. Anti-maleimide linker immune response has been detected where maleimide derivatives
containing certain chemical groups associated with the maleimide function were used, eg,
4-(pmaleimidophenyl)butyrate (MPB), m-maleimidobenzoate (MBS), or
4-(N-maleimidomethyl)cyclohexane-l-carboxylate (MCC) [14–16]. In this study, we used (simple) maleimides (present
in DSPE-PEG2000-Maleimide and NEM) in preference to maleimide derivatives, to avoid
immunogenic/toxic side-effects due to the presence of additional/unnecessary chemical groups
associated with the maleimide linker. In clinical translation, conjugate products containing
maleimide or even maleimide derivative (o-phenylenedimaleimide) such as the Hemospan (a
conjugated haemoglobin (MP4) blood substitute)  or the conjugated saporin (used in
cancer treatment) , respectively, have been tested in humans without significant
immunogenic/toxic side-effects attributable to the maleimide linker per se. Hemospan has completed
Fig 7. Real-Time Ultrasound Molecular Imaging of Esel Expression in the Mouse Abdomen. Combined 14MHz ‘CPS-contrast only’ and ‘B-mode
images’ before (i, ii, v, vi) and 30min post (iii, iv, vii, viii) bubble administration. ‘CPS-contrast only’ = bubble signal intensity in heated object scale. ‘B-mode’
= tissue signal intensity in grey scale. Arrows indicate the kidneys (K), liver (L), or spleen (S).
Phase 3 clinical trial . Nonetheless, formal immunogenicity and toxicity studies of our
bubble will be required. The thioether bond formed between maleimide and thiol is strong and
rapid at near neutral pH (bond strength in the order of nanonewtons; second-order rate
constant 0.8–1.2x104 M-1s-1) . The near neutral pH is advantageous in avoiding negative
impact on the ligands and bubbles during the conjugation process, and preventing dissociation
of the ligands from the bubbles in vivo.
The lipid component containing the conjugation functional group generally makes up 1–5
mol % of the bubble composition, range 1–20 mol % (Table A in S1 File). Our bubble falls into
Fig 8. Acoustic Quantification of Esel Expression. Acoustic signal intensity of targeted bubbles retained in the WT myocardium (R24) correlated with the
level of Esel expression. WT (blue); KO (red); not applicable (NA). R24 is plotted as mean with SD (error bars). Pearson r (r) is shown. WT mean (SEM,
range) = 0.32 (0.11, 0.01–1.07) AU, n = 12. KO mean (SEM, range) = 0.05 (0.02, 0–0.14) AU, n = 8.
the mid high range with 9 mol%, giving an estimated 30k/μm2 bubble surface area or 400k/
bubble of maleimide functional groups available for linking with targeting ligands (calculation
on page 7 of S1 File). The average of quoted targeting ligand densities in the literature is 5–30k/
μm2 bubble surface area (range 0.6–86 k/μm2) or 100–400k/bubble (range 3–820 k/bubble),
Table A in S1 File. Thus, our bubble contained enough maleimide groups for conjugating a
sufficient number of targeting ligands onto the bubble surface for efficient target binding under
flow conditions, as demonstrated in this study. However, it should be noted that amongst
various factors, a bubble’s target binding efficiency is also dependent on the targeting ligand’s
binding kinetics and valency.
The conjugation reaction ratio used to produce the successful Esel targeting bubble in this
study was 4x106 F(ab’)2 molecules per bubble, the estimated F(ab’)2:maleimide reaction ratio
was at least 10:1 (and each F(ab’)2 contained 2 thiol groups). We have deferred measuring the
actual number of F(ab’)2 grafted per bubble, because a robust methodology for this is currently
lacking. However, with a 10 molar excess of F(ab’)2 relative to the maleimide functions, we
expect the number of F(ab’)2 on a bubble to be roughly the same as the number of maleimide
functions on the bubble, ie, 30k/μm2 bubble surface area or 400k/bubble. This assumes
saturation of all maleimide functions, each linked to one F(ab’)2 molecule. Lower F(ab’)2:bubble
reaction ratio of 1x106:1 ( 2.5 molar excess of F(ab’)2 relative to the maleimide function)
produced bubbles that could only attach to target under static conditions in vitro , presumably
due to insufficient targeting ligand density achieved on the bubble-shell. The 10 molar excess
of F(ab’)2 compares favorably with the 10  or 30 [21, 22] molar excess employed by others
in similar maleimide-thiol bubble conjugation reactions. BR55, a targeting bubble being tested
for clinical translation by the Bracco pharmaceutical company [23, 24], used a 1:1 molar ratio
in the conjugation reaction between its targeting ligand and free lipid containing the
conjugation function, prior to bubble formation . It is important to note that in general, 10% of
the total shell components is incorporated into the bubble-shell (although the component
molar ratio remains essentially unchanged) . Therefore 90% of the targeting ligand is lost
when using the pre-bubble conjugation strategy. Perhaps significantly the amount of targeting
ligand lost is not dissimilar to the 10 molar excess used in the conjugation strategy reported
here. Thus, the use of 10 molar excess targeting ligands relative to the maleimide function in
the conjugation reaction should not make translation economically non-feasible. However,
future studies would be desirable to: (1) determine the minimum excess of targeting ligands
required to saturate all active (potentially toxic) maleimide functions on the bubbles; and (2)
compare the measured number of targeting ligands bound per bubble with that predicted.
The use of F(ab’)2 instead of whole mAb as targeting ligands eliminated any Fc-medicated
non-specific interaction or immunogenic side-effects. The translatability of a bubble may
depend more on the bubble’s generic composition and conjugation chemistry than the
targeting ligand per se, because the latter can be exchanged for one that is human-compatible and
binds efficiently to the molecule of interest without significantly altering the bubble’s other
properties important for clinical applications (eg, in vivo stability and acoustic response
favourable for real-time and quantitative imaging; low degrees of non-specific binding/retention in
multiple tissues favourable for broad applications; non-toxic/biocompatible). It remains to be
determined whether or not the generic composition of our bubble (with minor modifications
outlined in limitations below) can form the basis for generating a series of targeting bubbles
against various molecules for potential clinical applications in the future.
Our bubbles were frozen for long-term storage. Common frozen products widely used in
clinical practice include blood products such as the fresh frozen plasma and cryoprecipitate,
and cells used in bone marrow transplantation or stem cell therapy. Although frozen
formulations are feasible for clinical use, non-frozen formulations would be preferred for logistic
reasons (easier transportation and long-term storage). Non-frozen bubble formulations include
as a lyophilisate (dry powder) or aqueous lipid dispersion (solution) in a sealed vial containing
the bubble gas, where the bubbles can be reconstituted by the bedside just prior to use [27–29].
However, they require extra steps (time consuming) and do not offer clear advantages in our
early stage of bubble development. All three storage formulations are prone to generating
fragments that compete against the bubbles for attachment to molecular targets. In the non-frozen
formulations, some investigators re-wash the reconstituted bubbles before use , while
others do not [30, 31]–washing inevitably leads to a degree of bubble loss. In our study, the once
thawed targeting bubbles (without re-wash) worked well in practice. Their efficacy in
ultrasound molecular imaging and quantification suggested no significant negative impact from the
once freeze-thaw cycle or bubble fragments. Comparative studies to determine the amount of
target competing fragments generated in the single freeze-thaw process used in the present
study, as compared to bubble reconstitution from lyophilised powders (eg, BR55 used in Phase
0–2 clinical trials [23–25, 32]), effect on bubble targeting, and the threshold beyond which they
would cause significant negative impact on molecular imaging is useful, and remains to be
Outside of our work, as far as we are aware from publications in the English language of
phospholipid-shelled targeting microbubbles that have been tested in vivo for ultrasound
molecular imaging (Table A in S1 File):
1. Eight bubbles based on maleimide-thiol conjugation chemistry have been reported. These
bubbles differed from ours in terms of their generic composition, including the gas (eg,
C4F10 [20, 22, 30, 31, 33–35] instead of C3F8), the use of maleimide derivatives (eg, MPB)
[36, 37] instead of maleimide, or the number/type/combination/molar ratio of the shell
components. Their general properties are therefore likely to differ from ours. Only three of
the eight bubbles showed their effectiveness for ultrasound molecular imaging in more than
one non-reticuloendothelial tissues [22, 30, 31, 34, 35, 38, 39]; none showed effectiveness for
acoustic quantification of the targeted molecule to a high degree of quantification, Table A
in S1 File.
2. One bubble was recently reported for ultrasound molecular imaging mono-specific for Esel,
in ischemia-reperfusion injury of the heart , and in LPS-induced inflammation of the
hind-limb muscle  in rats. However, unlike our bubble, it contained immunogenic
streptavidin and its effectiveness for acoustic quantification of the targeted molecule to a
high degree of quantification was not demonstrated, Table A in S1 File.
3. Four bubbles targeting selectins (not mono-specific for E-, P- or L-selectin) have been
reported for the imaging of inflammation in the heart [31, 34, 40, 41], skeletal muscle ,
large bowel  or tumor  in animals. Unlike our targeting bubble which is
mono-specific for Esel, they are not suitable for the specific detection of endothelial activation which
starts early in inflammation. This is because P-selectin is expressed on both platelets and
endothelial cells, while L-selectin is expressed on lymphocytes. Biotin-streptavidin linkage
was used in three of these bubbles [33, 40–42], and maleimide-thiol linkage in the other [30,
31, 34] for grafting of targeting ligands to the bubbles, Table A in S1 File.
Elsewhere, ultrasound molecular imaging using targeting microbubbles mono-specific for
Esel has recently been reported in ischemia-reperfusion injury of the heart in rats  and in
tumors in mice . The bubbles used in these studies differ to ours in several major aspects,
eg, they are not phospholipid-shelled microbubbles and the conjugation chemistry used is
different. One used a double-shelled albumin (outer shell)—poly-DL-lactide (inner shell)
microbubble, grafted with targeting ligands using biotin-streptavidin conjugation chemistry ; the
other used a poly n-butylcyanoacrylate-shelled microbubble grafted with targeting ligands
using carbodiimide conjugation chemistry . In both cases the bubbles effectiveness for
realtime ultrasound molecular imaging and acoustic quantification of the targeted molecule were
Esel is an endothelial adhesion molecule, classically expressed only on activated endothelial
cells (basal expression is lacking and expression is absent in other cell types) . It can
therefore be used for the specific detection of endothelial activation, which occurs early in
inflammation . The classical vascular bed for Esel expression is the post-capillary venules .
However, it has also been found in the capillaries of the heart (in myocarditis)  and the
glomeruli (in glomerulonephritis)  in mice, similar to our observations in LPS-induced
inflammation of these tissues.
The mouse model of LPS-induced systemic inflammation was used in this study for imaging
Esel expression in inflammation. Widespread Esel expression in multiple organs was produced,
which allowed testing of our bubble for ultrasound molecular imaging in more than one tissue
in the same animal in one setting. It is a recognised model of endotoxemia and inflammation;
it reflects myocarditis or glomerulonephritis where widespread inflammation of the heart or
kidney occurs, respectively [47, 48]. In contrast to the models of ischemia-reperfusion injury or
transplant rejection, commonly used in this field for imaging inflammation, the LPS mouse
model is relatively easy to generate as it does not require surgery. Furthermore, surgical trauma
may confound analysis. The essentially global uniform expression of Esel in the heart in this
model (and Esel being expressed only on activated endothelial cells) was advantageous for
assessing the bubbles effectiveness for acoustic quantification of the targeted molecule, and for
investigating the cause of localised signal attenuations in the heart. Future studies applying the
bubble technology to other models of clinical disease would be desirable.
Ultrasound detection of Esel in the heart was limited by attenuations from the overlying
bone/air and retained bubbles located proximally in the ultrasound path. However, such
attenuations were overcome by using lower frequency ultrasound (greater penetrative depth) or
different imaging plane/angle. From the human imaging perspective, where the use of lower
frequency ultrasound (eg, 3–7MHz) and multi-plane imaging are the norm, and the footprint
of the transducer is much smaller relative to the body size (making it easier to achieve optimal
probe position/angle and avoid overlying bone/air), these attenuation issues are likely less
The in vivo specificity of our Esel targeting bubble was demonstrated using Esel KO mice as
negative controls. The KOs were used appropriately for this purpose for several reasons. They
were derived from the same genetic background and treated in the same way with
inflammation inducing agent as the WT animals; its local shear against bubble attachment/retention was
not significantly different from the WTs. Inflammation occurred in both the WT and KO
animals, even though Esel was absent in the latter. Although the number of adherent and
transmigrated leukocytes was higher in the WT animals , the retained bubbles were not seen
attached or phagocytosed by the leukocytes, suggesting that bubble retention in the WT was
essentially due to specific bubble attachment to Esel with little/no contribution from
non-specific bubble-leukocyte interactions. Confocal microscopy further confirmed specificity of the
bubble by demonstrating co-localization of the retained/attached bubbles with Esel expressed
on the endothelial cell surface. The use of the KO mice as negative controls, in preference to
using control bubbles conjugated with non-binding F(ab’)2, had the advantage of testing the
Esel targeting bubbles themselves rather than inferring about them from testing of the control
bubbles. It also avoided confounding due to differences in signal intensities from non-identical
size distribution between the targeting and control bubbles. Furthermore, non-binding ligands
on the control bubbles may well not be inert and hence may have their own contributions. An
alternative negative control in which Esel is blocked with free targeting ligand of the bubble
prior to bubble administration in vivo can be used. However this has its own limitations and
was not deemed necessary given the extent of proof already obtained in this feasibility study.
In general, the bubble composition and processing conditions are important determinants of a
bubble’s effectiveness for in vivo imaging applications, because they affect the bubble’s: (i)
halflife (stability) and acoustic response (signal intensity), relevant for real-time and quantitative
imaging (stable, echogenic, low attenuation bubbles with a relatively large linear range of
bubble concentration vs signal intensity are preferred); and (ii) degree of non-specific binding/
retention and toxicity (biocompatibility). These through their impact on the bubble size
distribution, shell properties (eg, gas diffusivity, thickness, surface tension, viscoelasticity, charge
and PEG content) and interactions with the host . The mechanisms are not well understood
and is an area of ongoing research beyond the scope of the present study.
The bubble dose used for ultrasound imaging was relatively large (108 bubbles; 5x109
bubbles/kg). This is approximately 10-fold the maximum dose of Sonovue (an FDA approved
nontargeting bubble) tested in humans without causing adverse effects. The standard clinical dose
for Sonovue (0.03ml/kg) is 0.006–0.015x109 bubbles/kg ( 0.4-1x109 bubbles for a 70kg man,
assuming a bubble concentration of 2-5x108/ml); the maximum dose tested was 56ml
(13x1010 bubbles or 0.16–0.4x109 bubbles/kg) . There is no standard dose of targeting
bubbles used in ultrasound molecular imaging. In mice, dosages used ranged 8x105–2x108 bubbles
( 0.03-8x109 bubbles/kg), Table A in S1 File. Like other investigators [8, 50–52] we did not
observe significant acute adverse effects using such a large dose of bubbles. Nonetheless, the
safety of such dosage in humans is uncertain and caution is needed. Hu et al , using a dose
of 108 targeting bubbles in mice, reported that high power bubble destruction may induce
transient reduction in blood flow in the targeted tissue region; the effect was reduced using lower
ultrasound power. In our study, high power bubble destruction was not used. The purpose of
the large dosage used in our study was two fold: (a) To provide excess bubbles to cover the full
range of molecular expression being quantified in the heart, and account for bubble ‘steal’ by
multiple tissues expression Esel. In man or other clinically relevant disease models, a smaller
dose (relative to body weight) may be sufficient because the number of tissues and peak level of
Esel expression are likely to be lower than that observed in the LPS mouse model used. We did
not carry out a formal dose titration study to confirm that the bubble dose used was optimal,
which would be useful as a future study. Nevertheless, the good correlation of R24 with Esel
throughout the expression range imaged suggested that the dosage used was adequate. (b) To
allow more sensitivity detection of non-specific bubble retention. Our bubble appears to have
one of the lowest degrees of non-specific retention compared with others. How much of this is
due to differences in the disease model or vascular bed imaged, or ultrasound setting used is
uncertain. Non-specific bubble retention may increase in the context of inflammation (due to
increased leukocyte presence) and in low shear flow regions (due to increased chance of
sustained bond formation and stasis eg, in the tumor neovasculature with blind ending loops).
Different ultrasound settings (mode, power, gain) will have different sensitivities for bubble
detection. We used low power CPS mode of a popular clinical ultrasound scanner for sensitive
real-time bubble detection. In our hands, the bubble showed low degrees of non-specific
retention in low shear flow regions of the heart, kidneys (and cremaster), in the context of
inflammation and high dose bubble administration. Future studies targeting different disease
processes, vascular beds and hosts will be required.
Formal immunogenicity and toxicity assessment of our targeting bubble was not performed.
However, potentially toxic active (unreacted) maleimide groups remaining on the targeting
bubble surface were minimised/eliminated by using excess targeting ligands in the bubble
conjugation reaction. And potentially toxic residual thiols on the targeting ligands on the bubble
were then inactivated using excess NEM, and unreacted NEM subsequently removed by
washpurification of the bubble. The potential immunogenicity/toxicity of the reacted maleimide
on the bubble (including reacted NEM) was discussed previously. The resulting targeting
bubble did not cause acute adverse effects in mice, but its medium- and long-term effects are
unknown. Before testing in humans, minor modification of the bubble will be required (such
as excluding DiI and using human-compatible targeting ligands) followed by formal
immunogenicity and toxicity studies in vitro and in animals. To date, BR55, a VEGFR2 targeting bubble
for cancer detection , different in composition and conjugation chemistry to ours (Table A
in S1 File), is the only targeting bubble tested in humans (Phase-0-2 clinical trial in prostate
cancer) [23, 24]–the results are still awaited. Notably, BR55 appeared to show moderate degrees
of non-specific retention in the healthy tissue (rat prostate) .
Targeting microbubbles for ultrasound molecular imaging, based on maleimide-thiol
conjugation chemistry with the generic (non-targeting components) composition described, may
possess properties (i)-(iii) desired for clinical applications.
S1 Video. Intravital Microscopy of Esel targeting microbubbles in an IL-1β pre-treated WT
mouse. Shortened movie clip containing CCD (bright field) and SIT (fluorescence) images. A
vein and artery can be seen running in a 5 to 11 and 7 to 3 o’clock direction, respectively.
Bubbles were administered via the tail vein at 14:30:15.
S2 Video. Intravital Microscopy of Esel targeting microbubbles in an IL-1β pre-treated Esel
KO mouse. Shortened movie clip containing CCD (bright field) and SIT (fluorescence) images.
A vein and artery can be seen running in a 6 to 1 and 9 to 3 o’clock direction, respectively.
Bubbles were administered via the tail vein at 12:58:35.
Drs Christoph Scheiermann, Mathieu-Benoit Voisin and Abigail Woodfin (Queen Mary,
University of London) for assistance in intravital and confocal microscopy. Dr Joseph Boyle
(Hammersmith Hospital) for assistance in immunohistochemistry. Profs David Cosgrove, late
Martin Blomley, Justin Mason (Imperial College London) and Mr David Dawson
(Hammersmith Hospital) for scientific discussions. Profs Sanjiv Kaul, Jonathan Lindner, Drs Alexander
Klibanov, Jiri Sklenar (University of Virginia) for JY’s training in bubble targeting (2004). Drs
Helene Houle, Pavlos Moustakidis (Siemens Medical Solutions, California) for technical
support with the Acuson Sequoia 512 (2003–8). Mrs Suzanne Schiller-Yeh for manuscript
Conceived and designed the experiments: JY CAS SN JMS DOH PN. Performed the
experiments: JY CAS EM. Analyzed the data: JY CAS EM RE MXT SN JMS DOH PN. Contributed
reagents/materials/analysis tools: JY CAS EM RE SN JMS DOH PN. Wrote the paper: JY CAS
EM RE MXT SN JMS DOH PN.
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