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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. 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