Download red blood cells as an ultrasound contrast agent

RED BLOOD CELLS AS AN ULTRASOUND
CONTRAST AGENT
Ali H Dhanaliwala∗ , Alexander L Klibanov† , John A Hossack∗
∗ Department
of Biomedical Engineering, † Department of Medicine - Cardiovascular Division
University of Virginia, Charlottesville, VA, 22903
Abstract—Microbubbles, shell-stabilized micrometersized gas bubbles, are the most common ultrasound contrast agent. Microbubbles can both improve ultrasound
image contrast as well as enhance drug delivery. Current
microbubble formulations, however, have a limited halflife in vivo and a limited therapeutic carrying capacity.
Red blood cells continue to be investigated as a drug
delivery vehicle given their large carrying capacity and
inherent biocompatibility. Red blood cells, however, cannot
be tracked in vivo and drug release cannot be specifically
triggered in space or time. In this paper, we propose a
novel ultrasound contrast agent that combines the benefits
of microbubbles with those of red blood cells. A method for
incorporating microbubbles into red blood cells to produce
acoustically active red blood cells (aaRBCs) is presented.
The acoustic properties and drug delivery potential of
these particles are investigated. B-mode ultrasound image
intensity of aaRBCs was 20 dB brighter than whole
blood. Release of a model drug from aaRBCs following
ultrasound insonation was 14-fold higher than controlloaded red blood cell ghosts.
Index Terms—Microbubbles, Red Blood Cells, Ultrasound Contrast, Drug Delivery
I. I NTRODUCTION
Microbubbles are gas bubbles, typically less than
10 µm in diameter to minimize the risk of emboli
formation, that are stabilized with a protein, polymer
or lipid shell [1] and injected intravenously to
enhance ultrasound image contrast. Microbubbles
are widely used as ultrasound contrast agents as
the acoustic impedance mismatch between the microbubble gas core and surrounding liquid strongly
reflects acoustic energy [3]. In addition, the oscillations of microbubbles in an acoustic field produce nonlinear energy (i.e. harmonics and subharmonics), which can be filtered to separate the
microbubble from background tissue signal [4],
further enhancing image contrast. Microbubbles are
used in several clinical applications and can assist
in the acquisition of a clinically useful image from a
“difficult to image” patient [5]. Applications include
left ventricular opacification to highlight cardiac
wall motion defects [6], and a burst-refill technique
to determine organ perfusion rates [7].
In addition to imaging applications, microbubbles
are also being investigated for therapeutic applications. Microbubbles insonated in close proximity to
cells have been shown to cause enhanced uptake of
drugs or genes through a process termed sonoporation in which the microbubbles create transient holes
in the cell membrane [8]. To localize the therapeutic
delivery, the drug or gene is attached to or dissolved
into the microbubbles [10]. Microbubbles have also
been shown to enhance tumor ablation [11] and
enhance sonothrombolysis [12].
Microbubbles have two main limitations. First,
microbubbles have a short lifetime in vivo. Once
injected, microbubbles are subject to filtration by
the liver [13], lungs [14], and spleen [15]. As a
result, microbubble clearance can occur as quickly
as five minutes after administration [17]. Consequently, high concentrations (109 [18]) need to be
administered in order to ensure that an adequate
number of microbubbles reach the desired target.
In addition, physiological phenomena that may take
hours to develop, such as brain edema [19] could
not be imaged continuously with microbubbles and
ultrasound. Second, microbubbles have a limited
payload. The microbubble shell layer has a limited
volume (a 1 µm diameter microbubble with a 10
nm thick shell has a total volume of 0.1 fL) and
can only incorporate hydrophobic drugs. Plasmids
can be attached electrostatically, but are limited by
shell surface area and must utilize positively charged
lipids that are not as biocompatible. Alternatively,
drug-containing liposomes [20] could be attached to
the microbubble shell, or the shell could be made
thicker in order to increase loading capacity [21];
however, both these techniques introduce new limitations. New microbubble preparations to improve
circulation life-time, improve biocompatibility, and
increase therapeutic payload continue to be pursued.
Red blood cells are similar in size to microbubbles, are produced at a rate of two million per
second, and continue to be investigated as a platform
for drug, gene, and enzyme delivery [22]. Red blood
cells are ideal drug delivery vehicles as they are
inherently biocompatible, have a long half-life (up
to a month [23]), and have a large therapeutic
carrying capacity (a human red blood cell has an
internal volume of 90 fL). In the most common
implementation, red blood cells are lysed then resealed in the presence of the desired therapeutic,
resulting in the loading of the desired molecule into
the cytoplasm. Due to the loss of hemoglobin, red
blood cells that have been lysed and resealed become pale and thus are termed “ghosts”. Targeting,
or sensitizing moieties, can be added to enhance
targeting or provide a photo or pH sensitive trigger
for therapeutic release [24].
Red blood cell ghosts as a drug delivery vehicle
have their own limitations. Once administered, red
blood cell ghosts are difficult to track in real-time
in vivo. Unlike microbubbles, at clinical ultrasound
imaging frequencies (6 – 15 MHz), red blood
cells do not provide sufficient acoustic impedance
mismatch and are therefore effectively invisible to
ultrasound [25]. Drug release from red blood cell
ghosts is also difficult to trigger. Light has limited
depth penetration and pH is difficult to control in
vivo. As a result, therapeutic release is dictated by
diffusion out of the cytoplasm.
We propose a new ultrasound contrast agent,
termed acoustically active red blood cell ghosts
(aaRBCs), that combines the advantages of microbubbles to offset the limitations of red blood cell
ghosts and vice versa. In this framework, microbubbles are incorporated into red blood cells to produce
aaRBCs. The microbubbles inside the red blood cell
will enable ultrasound tracking of the red blood cells
in real-time in vivo, as well as provide an acoustic
trigger for release of the loaded therapeutic. The red
blood cell will provide a biocompatible membrane
to protect the microbubble and increase circulation
Dhanaliwala
Reseal
& Wash
Lyse
RBC
+
+/-
Microbubbles
aaRBCs
Drug
Fig. 1. Procedure for loading microbubbles into red blood cells.
Washed red blood cells are placed in a hypotonic solution containing
microbubbles. Concentrated saline is then added to restore isotonicity,
and the solution is incubated at 37 ◦ C to facilitate membrane resealing. The cells are washed to remove unencapsulated microbubbles and
the resulting in acoustically active red blood cell ghosts (aaRBCs)
time, as well as a larger therapeutic carrying capacity. In this paper, we outline the production of these
new particles and provide initial characterization of
the acoustic response and triggered drug release.
II. M ETHODS
A. Acoustically-active red blood cell production
Acoustically-active red blood cell ghosts (aaRBCs) were produced using a modified dilution lysis
method [26] (Fig. 1). Briefly, human blood from
the University of Virginia clinical laboratory (IRB
waiver on file), collected in 1.8 mg/mL ethylenediaminetetraacetic acid (EDTA, Sigma Aldrich, St.
Louis, MO) to prevent clotting, was acquired and
used within 7 days. The whole blood was washed
three times in isotonic PBS (155mM, pH 7.4, Sigma
Aldrich, St. Louis, MO), removing the plasma
and buffy coat, then stored at 4 ◦ C. To lyse
the red blood cells, 1 vol. of packed red blood
cells was added to 40 vol. of lysing media. The
lysing solution consisted of 2.5 mM saline containing lipid-shelled microbubbles [27]. The fluorophore 1,1-Dioctadecyl-3,3,3,3 tetramethylindocarbocyanine perchlorate (DiI, Ex/Em 549/565, Invitrogen, Grand Island, NY) was added to the microbubble lipid shell to enable fluorescence microscopy
confirmation of microbubble loading. After adding
the lysing media, a sufficient volume of a 5 M
saline solution was added to restore isotonicity.
To facilitate resealing of the membrane, the cells
were then incubated for 60 minutes at 37 ◦ C. The
microbubble loaded red blood cell ghosts were then
washed in isotonic saline until the supernatant was
free of unencapsulated microbubbles. The resulting
2
aaRBCs were stored in isotonic saline and used
within 24 hours. Control red blood cell ghosts were
produced in a similar fashion except microbubbles
were omitted from the lysing media. The aaRBCs
were imaged using phase contrast and fluorescence
microscopy to confirm loading. Loading efficiency
was determined by optically counting the number
of red blood cell ghosts containing microbubbles.
A Coulter counter (Z2 Particle Analyzer, Beckman
Coulter, Brea, CA) was used to determine concentration and size.
B. Acoustic Characterization
Microbubbles, control red blood cell ghosts, or
aaRBCs were diluted in whole blood to a concentration of 150 × 106 particles/mL and pulled
through a 200 µm inner diameter cellulose tube
placed at the focus of a 2.25 MHz, 12.7 mm
diameter, ultrasound transducer (Panametrics M306,
Olympus, Waltham, MA). The tube containing the
sample was insonated with a 32 µJ pulse at a
pulse repetition frequency (PRF) of 200 Hz using a computer controlled pulser/receiver receiver
(Panametrics 5900PR, Olympus, Waltham, MA). A
bandpass filter between 1 20 MHz and a 26 dB gain
were applied to the signal using the pulser/receiver.
The received acoustic signal was captured using
a 14 bit A/D capture card (Compuscope 14200,
GaGe, Lockport, IL) at a sampling frequency of
80 MHz. The signal was then averaged (n = 5000)
and the background acoustic signal of the tube
was subtracted using MATLAB (Mathworks Inc.,
Natick, MA).
To determine the acoustic response when imaged
with a clinical scanner, microbubbles, whole blood,
or aaRBCs were placed in an acoustically and optically transparent cassette (NUNC Opticell, Thermo
Fisher Scientific, Waltham, MA) and imaged using
an Acuson Sequoia 512 ultrasound scanner and a
15L8 transducer (Siemens, Mountain View, CA).
Both B-Mode and a contrast specific mode (CPS
[4]) were used to acquire images of the particles. An
image-intensity versus microbubble concentration
calibration curve was acquired and used to convert
image intensity into microbubble concentration.
To determine the acoustic response when imaged
with an intravascular ultrasound (IVUS) system, microbubbles, control red blood cell ghosts, or aaRBCs
Dhanaliwala
Fig. 2. The four types of aaRBCs: (A) microbubble attached to the
outer membrane of the red blood cell ghost; (B) microbubble wholly
contained within the red blood cell ghost cytoplasm (C) microbubble
attached to the inner leaflet of the microbubble ghost and (D) empty
red blood cell ghost containing no microbubble. Arrows point to DiIlabeled microbubble. Scale bar = 10 µm
were diluted in whole blood and imaged using a
an In-Vision Gold ultrasound system and the 45
MHz Revolution intravascular ultrasound catheter
(Volcano Inc., San Diego, CA). The catheter was
placed in a 15 mL centrifuge tube containing the
sample, and B-mode images were acquired and
analyzed to determine the acoustic response of the
particles at high frequencies.
C. Microbubble Survival
The effect of centrifugation speed on microbubble
longevity was investigated to determine the effect
of the washing steps on the microbubbles. Microbubbles in saline were centrifuged at 1,000x,
3,000x, and 10,000x relative centrifugation force
(RCF) and the concentration was measured with a
Coulter counter (Multisizer III, Beckman Coulter
Brea, CA) every 20 minutes. The concentrations
were normalized to the initial pre-centrifugation
microbubble concentration and a curve of best fit
that maximized R2 was calculated.
D. Triggered release
The membrane-impermeable fluorophore calcein
(Ex/Em 470/509, Sigma Aldrich, St. Louis, MO)
3
Microbubbles
RBCs
Ghosts
aaRBCs
Calcein−aaRBCs
Normalized Distribution
1
0.8
0.6
0.4
0.2
0
1
2
3
4
5
Diameter (µm)
6
7
Fig. 3. Diameter distribution of microbubbles, red blood cells, red
blood cell ghosts, aaRBCs and calcein-loaded aaRBCs, as measured
by a Coulter counter
Fig. 4. Microbubble loss at centrifugation speed of 1000, 3000, and
10,000 RCF. Microbubble loss at 1000 and 3000 RCF were linear,
while microbubble loss at 10,000 RCF was best described by an
exponential function. (R2 > 0.98 for all fits)
was used as a model drug. To produce calceinloaded aaRBCs, 1mM calcein was added to the
lysing solution with microbubbles. The aaRBCs
were otherwise resealed and washed as above.
Control calcein-loaded red blood cell ghosts were
produced in a similar manner except microbubbles
were omitted from the lysing solution. To determine
whether ultrasound can trigger drug release, samples of calcein-loaded aaRBCs and control calceinloaded red blood cells ghosts were insonated using
a clinical ultrasound scanner (15L8 probe, Acuson
Sequoia scanner, Siemens, Mountain View, CA).
The samples (n = 3) were insonated for 10 minutes
at a frequency of 7 MHz and a mechanical index
(MI) of 1.9. The supernatant was sampled before
and after insonation. The cells were then lysed
with 0.1 % Triton X (Sigma Aldrich St. Louis,
MO) and the supernatant was sampled again to
determine total loaded calcein. The fluorescence
intensity of the supernatant samples was measured
with a fluorescence plate reader and used as a surrogate for calcein concentration. Drug release was
calculated as the difference between fluorescence
before and after insonation, normalized to the total
loaded calcein.
the red blood cell ghosts. Four types of red blood
cell ghosts were observed 1) microbubble attached
to the outer membrane of the red blood cell ghost; 2)
microbubble wholly contained within the red blood
cell ghost cytoplasm 3) microbubble attached to the
inner leaflet of the microbubble ghost and 4) empty
red blood cell ghost containing no microbubble
(Fig. 2 A-D). Only types 2 and 3 were classified as
aaRBCs. aaRBCs with microbubbles wholly contained within the red blood cell ghost were confirmed by acquiring time lapse images and verifying
microbubble motion within, but not outside, the red
blood cell ghost. It should be noted, however, that
current separation methods do not allow aaRBCs
to be separated from red blood cell ghosts with no
microbubbles or microbubbles attached to the outer
membrane. As a result, concentrations measured by
the Coulter counter include all four classifications.
Production efficiency of aaRBCs was 20 %. The
low efficiency is attributed to several factors. The
size of the pores in the red blood cell membrane
following lysing are at most 1 µm in diameter [51]
while the majority of the microbubbles are greater
than 1 µm in diameter (Fig. 3). Unfortunately,
a
limitation of the Coulter counter prevents the
III. R ESULTS AND D ISCUSSION
concentration of microbubbles with diameters less
A. Production of acoustically-active red blood cell than 1 µm from being measured. Second, during the
ghosts
lysis phase, the microbubbles must diffuse into the
Fluorescence images overlaid on bright field im- red blood cells; however, microbubbles preferential
ages confirmed the loading of microbubbles inside float up, potentially limiting diffusion. Finally cen-
Dhanaliwala
4
Fig. 5. B-mode image of (A) 106 /mL aaRBCs in an opticell using
an Acuson Sequoia 512 clinical scanner in CPS mode at 8 MHz, MI
= 0.2. (B) MI was increased to 1.9 for 20 s and aaRBCs were imaged
again, resulting in a decrease in image intensity. (C) Image of 106 /mL
plain microbubbles. (D) Image of whole blood. (E) Calibration curve
of microbubble concentration versus video intensity (R2 > 0.94).
106 /mL aaRBC had the same image intensity as 105 /mL MBs
Fig. 6. Top: Fluorescent images of calcein (green) and MBs (red)
overlaid on bright field images of red blood cells showing successful
production of calcein-loaded aaRBCs (image width = 10 µm). Bottom: Calcein-loaded aaRBCs showed a 14-fold increase in calcein
release as compared to calcein-loaded red blood cell ghosts (p <
0.001)
B. Acoustic Response
trifugation was used to separate aaRBCs from unencapsulated microbubbles. Microbubbles are typically not exposed to more than 100 RCF (relative
centrifugation force or g’s) as higher forces increase
gas diffusion out of the microbubble, ultimately
causing microbubble dissolution and collapse [28].
To adequately pellet the aaRBCs in a reasonable
time, higher centrifugation speeds are needed. While
microbubble loss is linear below 3000 RCF (R2 >
0.98 for all fits) (Fig. 4), 40 % of MBs were still
lost at the lowest RCF used to wash the aaRBCs.
As a result, microbubbles within aaRBCs may have
collapsed during the washing step.
Size characterization of control red blood cell
ghosts, aaRBCs and calcein-loaded aaRBCs demonstrated a reduction in size compared to packed red
blood cells (Fig. 3). During lysis, some of the
cell membrane may shear off prior to resealing
[29], resulting in reduced membrane surface area
and thus reduced cell volume. In addition, optical
observations suggest that that majority of the cells
obtain a spherical shape after resealing rather than
regaining their original biconcave discoid shape.
Dhanaliwala
When imaged with the single element transducer
at 2.25 MHz, aaRBCs exhibited an 8.3 dB increase
in signal over red blood cell ghosts, but a 4.4 dB
decrease in signal compared to plain microbubbles. aaRBCs had a similar frequency spectrum
as compared to plain microbubbles (Table I).The
differences in the spectrum may be due to damping
of microbubble oscillations by the cell membrane
[30] or may be due to the minimization of interactions between oscillating microbubbles that would
otherwise occur in a cloud of microbubbles [31]. On
B-mode images obtained from the clinical scanner,
image intensity of aaRBCs was 20 dB higher than
whole blood but 5 dB lower than microbubbles
(Fig. 5 A,C,D). Although the aaRBCs were concentration matched to the microbubbles using the
Coulter counter, the aaRBC sample actually contained a mix of aaRBCs and empty red blood cell
ghosts. As a result, it is unlikely that the aaRBC
sample contained the same number of acousticallyactive particles as the plain microbubble sample,
which may account for the reduced image intensity.
In addition to image intensity, the ability to rupture
aaRBCs was investigated by increasing the MI to
5
Particle
Fundamental
Second Harmonic
-6dB Bandwidth (MHz)
Peak (MHz)
Amplitude (dB)
Frequency (MHz)
aaRBC
1.63
2.64
-13
8.64
Microbubbles
2.67
2.8
-15
8.4
TABLE I
F REQUENCY CHARACTERIZATION OF THE RECEIVED MICROBUBBLE AND AA RBC SIGNAL FOLLOWING INSONATION AT 2.25 MH Z
1.9. After 20 seconds, the image intensity of the
aaRBCs was reduced (Fig. 5 B), suggesting the microbubbles inside the aaRBCs were being ruptured.
The number of aaRBCs in the sample was quantified acoustically using an acoustic calibration curve
(Fig. 5 E). A sample of aaRBCs at a concentration
of 106 /mL had the same image intensity as a sample
of microbubbles at a concentration of 105 /mL.
When imaged with the intravascular ultrasound
system at 45 MHz, the background signal from
whole blood was no longer negligible. No increase
in image contrast over background was observed
with the control red blood cell ghosts, while aaRBCs
resulted in a 6 dB increase in image contrast.
C. Triggered Release
Calcein was successfully co-loaded with microbubbles into red blood cell ghosts (ghosts?)
to produce calcein-loaded aaRBCs (Fig. 6 –
Top). Calcein-loaded aaRBCs exhibited the same
four classifications as standard aaRBCs. After insonation, a 14-fold increase in calcein release
was observed from calcein-loaded aaRBCs when
compared to control calcein-loaded red blood cell
ghosts. A maximum of 20 % of the total calcein
from the calcein-loaded aaRBCs was released. This
is, again, probably a consequence of the low microbubble loading efficiency into red blood cell
ghosts. The insonated calcein-loaded aaRBC sample
contained both aaRBCs and red blood cell ghosts,
both of which would contribute to the total amount
of calcein in the system; however, only the aaRBCs
would result in calcein release during insonation. As
a result, only a fraction of the total calcein amount
was released during insonation.
IV. C ONCLUSION
By loading microbubbles into red blood cells, we
produced a novel ultrasound contrast agent termed
Dhanaliwala
acoustically-active red blood cell ghosts (aaRBCs).
Currently, loading efficiency of microbubbles into
red blood cells is low and additional optimization
in both loading microbubbles into the red blood
cells and isolating successfully produced aaRBCs is
required. Acoustic characterization with a single element transducer, a clinical ultrasound scanner, and
an intravascular ultrasound catheter demonstrated
that aaRBCs maintain the acoustic contrast of microbubbles. In addition, ultrasound successfully, and
specifically, triggered drug release from calceinloaded aaRBCs. Together, this demonstrates the
promise of aaRBCs as a biocompatible ultrasound
contrast agent and an acoustically triggerable drug
delivery vehicle.
ACKNOWLEDGMENT
This work is supported in part by the National
Institutes of Health NIBIB grant HL090700 to ALK,
JAH and UVA Coulter Translational Research Grant
to ALK, JAH. AHD is supported by an American
Heart Association Predoctoral Fellowship and a Virginia Space Grant Consortium Graduate Fellowship.
AHD thanks Dr. Dorothy Haverstick for providing
blood samples. The content is solely the responsibility of the authors and does not necessarily represent
the official views of the NIH, AHA, or VGSC.
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