Preparation Optimization And Evaluation Of Long-circulation Lipid-encapsulated Ultrasound Contrast Agent "Zhifuxian"

BackgroundThe preparation and application of novel ultrasound contrast agents (UCA) have gained wide interests. The development of the second-generation ultrasound contrast agents, a highly concentrated microbubble suspension, had evolved diagnostic US from a routine scan mode into a contrast enhancement mode. This is the 3rd revolution in diagnostic ultrasound, which can match CT or MRI contrast enhancement. Microbubble imaging agents enhance visualization of blood flow and improve our sensitivity and specificity of diagnosis. Furthermore, potential of microbubbles applications in molecular imaging and drug or gene delivery had emerged, once binded with targeted ligand or loaded with specific gene or drug. Therefore, it was predicted that ultrasound contrast agent would become a routine in ultrasound diagnosis and may be involved in noninvasive ultrasound therapy. It is of great medical and economic values.Ultrasound contrast agents have been developed from an air-filled microbubbles to perfluorocarbon-filled microbubbles, from albumin-encapsulated microbubbles to lipid-shelled microbubbles. Since then, the desirable microbubbles were described as lipid or polymer encapsulated, submicron or nanometer in size, better stability and longer lifespan in circulation .Most of the technologies and patents were developed and dominated in US and Europe in early 1990s. Optison?, Definity?, ImageatTM and SonoVue? made by US and European companies are now sharing the world market of UCA. In China, UCA research was late and fell backwards. So far, no domestic ultrasound contrast agents had been permitted to be used in the market. SonoVue? is now the only agent approved and used in Chinese market, although it is costly and survives shortly in circulation.Zhifuxian is the first and patented perfluorocarbon-filled lipid microbubble developed in China. Based on the new lipid microbubbles technologies, Zhifuxian was creatively designed and prepared by new lipids configuration and preparation technics. It is featured with high microbubbles production ratio, significant contrast effects, low dosage and long survival time. Especially on the long survival time of more than 30 min in circulation, Zhifuxian is superior to almost all of the other agents, and this allows longer contrast scanning and more detailed observation of solid organs, like liver and kidney. As a preliminary product, Zhifuxian was only a protype made in lab. Many technologies need to be improved on reproducibility, stability and the related technics. Furthmore, studies on acoustic properties, application values, methodologies and safety should be taken into consideration .Objectives:1. Improvement and optimization of the lipids configuration, the preparation technics.2. Assessment of physicochemical and acoustic properties of Zhifuxian.3. Applications of Zhifuxian in routine contrast imaging, specific targeted imaging, drug targeted delivery based on microbubbles vector and acoustic cavitation.4. Safety consideration includes intravenous injection and acoustic cavitation bioeffects.Methods and protocols:1. Statistical analysis: One-way ANOVA test or even design test for impact factors of preparation.2. Physicochemical properties of the microbubbles: the microbubbles concentration, pH value, surface potential and size distribution analyzed by using laser particle size analysator, RC-3000 Coulter counter, light microscope, transmission electron microscope(SEM).3. Stability test: acceleration test.4. Anti-pressure test: the bubble stability under different acoustic pressures or mechanic indices in vitro using ultrasound scattering intensity.5. Real-time contrast enhancement test: imaging with low MI mode, acquisition of the contrast effects from normal liver, cardiac chambers, myocardium, and liver or kidney bearing VX2 cancer.6. Elasticity and non-linear properties: analyzed by broad-band spectrum in Church model formula. 7. Specific conjugation of targeted ligands: including RGDS-covalent and RGDS-attached lipid microbubbles evaluated by flow cytometry and fluorescent microscopy for binding ratio and stability.8. Preparation of Paclitaxel conjugated lipid-microububbles by lyophilization and RP-HPLC detection of the entrapment efficiency (EE) of Paclitaxel.9. Safety tests including acute toxicity, maximum tolerance dose, hemolysis, venors stimulation, pyrogen reaction.10. Bioeffects of acoustic cavitation: In vivo tissue bioeffects were induced by diagnositic ultrasound burst of Zhifuxian microbubbles as cavitation nuclei, Evans-blue (EB) dye was used as a permeability tracer of heart and brain, in order to find any tissue permeability changes during diagnostic ultrasound exposure.11. Impact of the coordinated effects of Zhifuxian and diagnositic ultrasound to the rat memory ability was measured by evaluating the ability of spatial memory.Results and conclusions:1. The Zhifuxian microbubbles preparation were greatly improved by the following processes, including hydration control of lipid suspension, optimization of suspension freeze-dry curve and using mechanical vibration instead of sonication. The results showed:①better production ratio;②ideal particle size distribution from micro-scale microbubbles into including nano-scale submicron microbubbles.③better anti -pressure properties;④better stability;⑤longer survival time in circulation.2. The viscoelastic properties resulted in GS = 10 MPa , d Se= 0.1%R0,μS = 1.49N ? S m2, and the resonance frequency happened at 2.3 MHz.3. Subharmonic signal was provocated from the lipid microbubbles under certain acoustic pressure. The development of the subharmonic undergone three stages, i.e. occurrence, growth and saturation, and that hysteresis appeared in descending ramp insonation. The stimulated subharmonic signal under certain pressure may indicate that the lipid microbubbles can be generated a better source of ultrasound imaging in future..4. As a novel submicron ultrasound contrast agent, Zhifuxian was able to opacify cardiac chambers and myocardium for more than an hour, bearing the name of a long circulation agent. The detection of VX2 cancer in liver and kidney, especially the tiny tumor less than 10mm, greatly increased. Because of the stability and chemical setting, the microbubble could be conjugated with specific ligands or drugs as a carrier. It can be served in targeted imaging and drug delivery purposes. Covalent bonding is a better way link targeted ligands than directly attach..5. Neither significantly haemolysis nor agglutination could be found in rabbit study impacted by high IV dose of Zhifuxian. No vascular excitation, pyrogen reaction, and median lethal dose indicates there's no death of mice IV 500 times than routine dose, and not any apparently paradoxical reaction and death in maximum tolerated dose, toxicity eaqual to sodium Chloride, accord with the drug safety evaluation criterion. Safe IV.6. No BBB or myocardial permeability change of mice happened after five times IV dose, even high-output intensity ultrasound insonation for 10 min.BBB can be opened 4h reversible and selectively after big-dose Zhifuxian IV combination high mechanism index diagnosis ultrasound irradiation without any nerve behavior or brain cell morphology changes.