Ultrasound Microbubble Delivery Targeting Intraplaque Neovascularization Inhibits Atherosclerotic Plaque in an APOE-deficient Mouse Model

Materials and Methods

Animal and atherosclerosis induction. Six wild-type and 27 apolipoprotein E-deficient (Apoe^−/−) C57BL6 mice were obtained from Changzhou CAVENS Laboratory Animal Co., Ltd. (Changzhou, P.R. China). All mice were male, 3-4 weeks old and were maintained in a HEPA-filtered environment at 24-25°C and humidity at 50-60%. The wild-type C57BL/6 mice were fed normal chow. The Apoe^−/− mice were fed a hypercholesterolemic diet (Opensource Animal Diets Co., Ltd., Changzhou, China) containing 1% cholesterol, 10% fat, 10% egg yolk power and 0.2% sodium cholate. All mice were maintained on their respective diets for 30 weeks. Three Apoe^−/− mice fed the hypercholesterolemic diet fed were then randomly selected and euthanized and their aortas processed for morphologic and histopathological examination in order to confirm development of atherosclerosis. Remaining animals were then treated as described below. Animal experiments were approved by the Animal Committee of Nanjing Origin Biosciences, P.R. China (OB 1607).

Microbubble preparation. Targestar-SA microbubble (Targeson Inc., San Diego, CA, USA; distributed in China by Origin Bioscience, Nanjing, P.R. China) was used as the basis for drug-loaded and targeted microbubbles in this study. Targestar-SA is an ultrasound contrast agent composed of a perfluorocarbon gas core encapsulated by a lipid shell. The outer shell is derivatized with streptavidin, which binds biotinylated ligands at a density of 80-220×10³ molecules per microbubble. The agents have a median diameter of approximately 2.0 μm. Two types of microbubbles were produced. ICAM1-targeted microbubbles (MBi) were prepared by incubating microbubbles with a biotinylated antibody to ICAM-1 (Abcam, Cambridge, MA, USA) at room temperature for 20 min at a ratio of 0.7 nmoles of the antibody per 10⁹ microbubbles. ICAM-targeted and Endostar-loaded therapeutic microbubbles (MBie) were prepared by coupling both biotinylated anti-ICAM-1 and Endostar (Simcere Pharmaceutical, Nanjing, P.R. China) to the outer shell of the same microbubble at a ratio of 0.35 nmoles of each targeting moiety per 10⁹ microbubbles. For fluorescence-activated cell sorting analysis of the prepared microbubbles, the biotinylated anti-ICAM1 and Endostar were labeled with fluorescent dyes fluorescein isothiocyanate and rhodamine, respectively. Unreacted anti-ICAM-1 and Endostar were removed from the microbubbles by centrifugal washing, per the manufacturer's recommended protocol. The presence and conjugation rate of anti-ICAM-1 and Endostar to the microbubble surface were examined by epifluorescence microscopy (Olympus BX53; Tokyo, Japan) and flow cytometry (BD Accuri C6). Microbubble-bound Endostar was measured by the BCA Protein Assay (Pierce, Rockford, lL, USA). Unconjugated Targestar-SA microbubbles were used directly from the vial without the addition of ligand.

Treatment. Six wild-type C57BL/6 mice fed with normal chow were assigned to group 1 as normal control. The remaining 24 Apoe^−/− mice were randomly divided into four groups of six mice and assigned to groups 2-5 after confirmation of atherosclerosis 30 weeks following a hypercholesterolemic diet. Group 2 mice were untreated and served as model control; group 3 mice received Endostar (25 mg/kg) by retro-orbital injection; group 4 mice received MBi; group 5 mice received MBie. Mice were anesthetized with ketamine, acepromazine and xylazine and then placed in a dorsal recumbency position on a heated stage. A dose of 50 μl (1×10⁸) microbubbles per mouse was administered by retro-orbital injection with a 27-gauge needle. UTMD was performed for groups 4 and 5 using a hand-held sonoporator (Sonitron 2000, Artison, OK, USA). This device consists of a piezoelectric transducer with a center frequency of 1 MHz. The transducer was placed above the ventral midline of the abdominal and thoracic cavities with ultrasound coupling gel. The settings were optimized as follows: duty cycle was 50%, treatment duration of 30 s, with acoustic intensity of 2 W/cm². The sonoporator cycled on and off in 30-s intervals to enable the microbubbles to fully re-perfuse the aortas after each 30-s cycle of UTMD. This process was repeated three times. All treatments and UTMD were repeated 48 h later with the same doses and settings.

Morphologic measurement. All animals were sacrificed 2 weeks after the final treatment. After euthanasia, mice were placed in dorsal recumbency, and a ventral midline incision was made to expose the abdominal and thoracic cavities. The aorta was perfused with phosphate-buffered saline (PBS) through the left ventricle of the heart. Next, under magnification, microscissors and fine-point forceps were used to remove the fat surrounding the three aortic arch branches (brachiocephalic, left common carotid, and left subclavian). The aorta was removed from its origin at the left ventricle to a few millimeters distal to its bifurcation into the common iliac arteries. The aorta was freed from surrounding vessels by making cuts at each of the three branches of the arch, the origin of the aorta at the heart, and distal to the aortic bifurcation into the common iliac arteries. The dissected aorta was then photographed. The areas of the entire dissected aorta and atherosclerotic plaques were measured with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). The percentage of en face plaque area was calculated for each animal.

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Figure 1.

Atherosclerotic plaques in an apolipoprotein E (APOE)-deficient mouse model of atherosclerosis. Representative images for A: gross appearance, and B: hematoxylin and eosin (H&E) and C: intercellular cell adhesion molecule-1 (ICAM-1) immunohistochemical staining of the aorta in normal control animals. Representative images for D: gross appearance, and E: H&E and F: ICAM-1 immunohistochemical staining of the aorta with atherosclerotic plaque in an APOE-deficient mouse model of atherosclerosis. Orange arrows indicate atherosclerotic plaques. Magnification: H&E 100×; immunohistochemical staining 400×.

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Figure 2.

Microbubble charactarization. A: Representative bright-light image of microbubbles. B: Representative fluorescence image of the microbubbles conjugated with rhodamine-labeled Endostar. C: Representative fluorescence image of microbubbles conjugated with fluorescein isothiocyanate-labeled antibody to intercellular cell adhesion molecule 1 (ICAM-1). Magnification: 400×. Flow cytometric analysis of: D: Unconjugated microbubbles (MB), E: ICAM-1-targeted microbubbles (MBi) and F: ICAM-1-targeted and endostar-loaded therapeutic microbubbles (MBie). G: Conjugation rate of MBi and MBie.

Histology and immunohistochemistry. One segment of the aorta was obtained from each mouse after aorta perfusion. The aorta was fixed in 10% formalin, dehydrated, paraffin-embedded, sectioned at 4 μm and stained with hematoxylin and eosin (H&E). One microscopic slide with three sections from the paraffin block was created for each animal. Histological changes were evaluated by a pathologist who was blinded to the experimental groups. For immunohistochemical analysis, the sections were washed three times with PBS for 5 min each, blot dried and then treated with 3% hydrogen peroxide for 30 min at room temperature to block endogenous-peroxidase activity. The sections were immersed in antigen-retrieval solution (citrate buffer, pH 6.0) for 10 min, followed by rinsing with PBS. After blocking with normal goat serum for 30 min at 37°C, sections were co-incubated with primary antibodies against CD31 (BD Biosciences, San Diego, CA, USA) or ICAM-1 (Abcam) (1:100 dilution in PBS) overnight and then with a peroxidase-conjugated anti-rabbit IgG secondary antibody (Biosynthesis Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature. Subsequently, the sections were incubated with 3,3-diaminobenzidine reagent (ZSGB-BIO, Beijing, P.R. China) for 10 min, counterstained with hematoxylin, dehydrated and mounted for microscopy. The slides were viewed at 100× or 400× magnification with brown-stained cells being identified as positively-stained cells. Expression levels were quantified by the average optical density (AOD) of the positively-stained cells in 5 fields/sample with Image-Pro Plus 6.0 software (Media Cybernetics).

Statistical analysis. Statistical analysis was performed using SPSS16.0 software (SPSS Inc., Chicago, IL, USA). All results are expressed as the mean±standard deviation (SD). Comparisons between two or multiple groups were made with the Student's t-test or ANOVA. Values of p<0.05 were considered significant.