Preliminary Study On Phospholipid-coated Microbubbles Preparation And Platelet Receptor Target Conjugation

Background:With the development of the ultrasound contrast agent and imaging technology, ultrasound contrast imaging tend towards clinic gradually. In the same time, more wildly applications in the treatment and diagnosis of the diseases are recognized gradually with the development of the drug and gene encapsulated microbubbles. The microbubbles produce nonlinearity resonance, which lead to the microbubbles disruption and sending out the energy. In the process the drug and gene encapsulated in the microbubbles maybe deliver to the specific site. The microbubbles can be used as a tool for target treatment by the mechanism. Phospholipid-coated microbubbles have a good histocompatibility and high gene transfect efficiency. The intrinsic property of the phospholipid-coated microbubbles made it easy to conjugate in the shell of the microbubbles. The bubbles maybe a useful effective carrier for target treatment. In hence, the study of phospholipid-coated microbubbles is becoming hotspot. Several kinds of phospholipid-coated microbubbles have been successful development in abroad, but little has been done in domestic in the field. Our group have developed the albumin coated microbubble named "dongguan parenteral solution" and "quanfuxian". The acoustic activity of these bubbles is prior to the abroad's in many ways. The success of albumin microbubbles promotes us to develop a new king of bubble-phospholipid-coated microbubbles. Molecular imaging is being pursed in all arenas of noninvasive medical imaging, initially with nuclear and magnetic resonance imaging. As the ultrasound, early MR contrast agent werenonspecific, provide temporary tumor or organ accumulation. Recently, people have develop successfully site-specific contrast agent that accumulate and enhance MR detection at specific sites. As the site-specific contrast agent of MR, phospholipid-coated microbubbles with ligand conjugation can accumulate in the pathological site. The technology will perhaps lead to realize the dream of no-wounded, cheaper, effective target diagnosis. Objects:1. To explore the methods of stable phospholipid-coated microbubbles preparation and develop a new kind of bubble.2. To explore the methods of ligand conjugation of the microbubble which target to platelet receptor GP b / a and discuss the possibility of molecular imaging of ultrasound.Methods:1. Liposome was prepared with cholesterol and lectithin and the phospholipid-coated microbubble prepared by sonicated the liposome solution dissolving with glucose at 110 watt energy. The stability of microbubbles were compare with the different condition: with or without methoxypoly(ethylene glycol)and different concentrations of in different times and temperatures.2. The efficacy of the homemade lipid contrast agent was evaluated by comparing with "dongguan parentarel solution" and "quanfuxian". Different doses (very high0.225m1/kg, high0.15ml/kg, middle0.l5ml/kg, low0.05ml/kg, "quanfuxian" only in low dose ) were inject in bolous in random order. Left heart opacification grades and numbers of endocardial border delineation segment observed after each contrast injection.3. PEG-RGDS were synthesized by RGDS(Arg-Gly-Asp-Ser) peptide and PEG. The target microbubbles which has specific affinity to platelet GP II b / a receptor were produced by liposome and RGDS solution after sonication. The effect of the target microbubbles affinity to microthrombi formed by platelet aggregation was investigated. In additional, the signalto noise ratio of the artificial thrombi was evaluated by two dimensionultrasound after target microbubbles injection in vitro. 4. Platelet GPb / a receptors were activated by ADP added to wholeblood. CD41 and CD61 were detected by FCM after target or controlmicrobubbles administered. The ability of target microbubbles affinity toplatelet GP II b / a receptors was discussed. Results:1. The concentrations and diameter of the phospholipid-coated microbubbles were 1.25 l0/ml and 3.745?.7m. With the increase...