The Preparation And Evaluation Of Lipid-coated Microbubbles Targeted To VEGF And α_vβ₃ Integrins

Background:With the development of ultrasound contrast agent and contrast specific imaging technology, contrast ultrasonography greatly improves the contrast resolution and promotes the sensitivity and specificity of ultrasound diagnosis. Now specific targeted contrast agent is becoming more and more interesting for its target imaging ability to specific receptors once conjugated with specific antibodies or ligands. Targeting microbubbles are possible to image thrombus, neovascular endothelium and tumor cells and have potential to bind drug or genes for therapeutic purposes.In the research of tumor growth, infiltration and metastasis, many specific markers of tumor cells or neovessel had been found. These makers could be expressed on neovascular endothelium or on tumor cells. Angiogenesis receptor are those markers which help to promote neovessel formation to meet the need of tumor growth. For many angiogenesis receptors located on neovascular endothelium, they became the target of anti-angiogenesis strategy. Some other specific tumor makers had been found on tumor cell membrane and had been used in early tumor diagnosis. Specific ultrasound imaging capable of targeting to neovessel formation would be possible and desirable if specific antibody or ligand could be conjugated on microbubbles. To achieve this, the initial and important step is to bind a special ligand to ultrasound contrast agent. Although the technique to prepare targeted ultrasound contrast agents had been described in literatures, many details and skills could not be found. So, basic preparation of the targeted ultrasound contrast microbubbles is necessary.Objective:This study was designed to find an effective method to prepare an ultrasound contrast agent specific targeted to tumor angiogenesis. The study included: evaluation of the physiochemical properties of "Zhifuxian", a new long persistent lipid-coated microbubbles ultrasound contrast agent made in our lab; specific conjugation of VEGF antibody oravpyintegrins antibody to the microbubbles; in vitro assessment of the targeting ability of the specific bound microbubbles.Method:Physiochemical properties: The microbubbles size and distribution were analyzed by Olympus BX50 optical microscope using the magnitude of xlOO and x400. The microbubbles concentration was detected by a Sysmex KX-21 blood cell multisizer. The PH value and Zeta potential were assessed by Zeta SIZER 3000 and the viscosity was analyzed by a viscosimeter.Conjugation of antibody to microbubbles: Anti vascular endothelial growth factor (VEGF) polyclonal antibody was diluted and was added to the lipid microbubbles, mixed, adjusted the PH value from 4 to 5, and incubated for 2 h at 4°C. Then the suspension was washed two times with 0.1 mol /L phosphate-buffered saline (PBS). The microbubbles targeted to av|33 integrins was prepared by the same method using 75 ug avP3 mono-colonial antibody.Fluorescent labeling of plain microbubbles: Dioctadecyloxacarbocyanine perchlorate (DiO), a fluorescence molecular probe, was dissolved in dimethylsulfoxide (DMSO) at different concentrations from 0.0025 to 0.25 mg/ml. Immunofluorescent assay was performed to detect the fluorescent brightness of DiO stain with different concentrations. The proper concentration was found by selecting the maximal dilution given out good fluorescent intensity. The DiO solution was then mixed with the lipids suspension for ultrasound contrast agent preparation. Sonication and mechanical vibration were used in the bubble-making procedure. 15 min after preparation, the final microbubbles suspension was washed twice with PBS and was checked under fluorescent microscope.Fluorescent Labeling of targeted microbubbles: The indirect immunofluorescent technique should be used to label the targeted microbubbles. Rhodamin-conjugated affinipure coat anti-rabbit IgG was added to the targeted microbubbles, after adjusting the PH value to isoelectronic point (PI) of secondary antibodies for 30 min at room temperature. The targeted microbubbles were then repeated washed two times with PBS buffers to elute any free antibody and fluorescence particles. Red shinning fluorescence was observed immediately after washing four times or after four weeks storage in 4°C.Targeting ability of the microbubbles with 0^3 integrins antibody: The targetingability of the avP3 integrins antibody conjugated microbubbles was tested in cultivated kl735 M2 melanin tumor cells. Results:1. The diameter distribution of Zhifuxian ranged from 3um to lOum with mean diameter of 4um, 98% of the bubbles less than 8 um. The bubbles concentration was 7><109 /ml, the PH value was 6.42 and the electronic potential in saline is -35.5mv. Zhiifuxian was consisted of lipids shell, perfluorocarbon gas core and suspension solution. The lipids have a hydrophilic end and a hydrophobic end, which is capable of binding any hydrophobic materials like DiO.2. The VEGF and avP3 integrins antibody conjugated on lipid microbubbles gave out bright red fluorescence in immunofluorescent assay after four times repeated washing. The microbubbles size and distribution remained unchanged, but the bubbles concentration dropped from 109 to 108. The bright red fluorescence remained strong one month after storage in 4°C.3. Both of sonication and mechanical vibration could bind the lipid microbubbles with DiO. The DiO-labeled microbubbles gave out bright green fluorescence when excited with 500nm light-wave, but the microbubbles made by sonication seemed to attach more DiO on the bubble film and resulting in green opacification.4. The microbubbles targeted to avf53 Integrins could actively adhere to the melanin tumor cells very well. A lot of the targeted microbubbles were found around the targeted cell. The number of the targeted bubbles attached to a single melanin tumor cell ranged from 3 to 9, while in the control group, few control bubbles could found.Conclusions:1. Zhifuxian, a new lipid-coated microbubbles, had been found to be biocompatible from its physiochemical aspects. Zhifuxian was negative charged and its electronic potential in saline was up to -35.5 mv. This made it possible to prepare the targeted microbubbles by using static adsorption.2. The conjugation of antibody to the microbubbles could be achieved by static absorption. The targeted microbubbles were physiochemically stable.3. The targeted microbubbles could be labeled with Rhdamine labeled IgG, shinning bright red fluorescence. DiO, a positive charged molecular probe, could be conjugated onthe bubbles film and be used as a fluorescent marker. Sonication seemed to attach more DiO to bubbles film than mechanical vibration.4. The specific binding of microbubbies with the ligands (anti-VEGF antibodies by the fluorescent secondary antibodies or avfb Integrins) could be identified.5. In vitro study, the targeted ability of the specific-bound microbubbies were able to adhere to the melanin tumor cells actively.