Preparation And Quality Research Of PH-sensitive Liposome

Liposome is a kind of close vesicle , which is consist of double lipid molecules with water inside. Its structure is more like biological membrane which has two characters of hydrophile and hydrophobicity after balancing in water. Liposome can wrap many kinds of materials in water phase and membrane phase. It is widely used in many fields, such as cell biology, immunology and genetic engineering. As disease mechanism is deeply known in molecule level , gene therapy has turned into an important task in the research of medical biology. Served as the carrier of outer genes, liposome has many merits, such as easy preparation, innocuity, no immunogenicity and resistant to nuclease. It wraps antisense oligonucleotides( ASODN) and transfers them to target cells effectively. Because pH -sensitive liposome has the structure that has sensitivity in pH changing, Its contained materials can be released in acid conditions of infective sites and tumorous tissues. According to the development, the author uses the superiority in our laboratory and works in preparation and quality research of pH-sensitive liposome.1. Preparation of pH-sensitive liposome, observation of conformation, determination of envelopment ratioAccording to literatures, the pH-sensitive liposome consisting of DOPE/OA/ Choi can be meld with cell membrane in acid conditions and has sensitivity in pH changing. We also observe its conformation and structure with super-microscope. Further the author use l25I-IL-8 as wrapped material to measure the envelopment ratio of liposome made by what kind of method through taking count of isotope . Because we know5that I-IL-8 and ASOND have near molecular weight, we can explore the best envelopment ratio of wrapping ASOND and research appliance of liposome in molecule level. Through using four kinds of methods: mechanical dispersion, calcium-induced fusion, reverse-phase evaporation , calcium-induced fusion with reverse-phase evaporation and measuring the liposome of envelopment ratio, we have the conclusion that combined calcium-induced fusion with reverse-phase evaporation has the highest envelopment ratio that is nearly 50%. Using this method, we establish the basic method for wrapping ANOSD in liposome.2.The biological activity of pH-sensitive liposome in cell (1). The analysis of pH sensitivityWe put pH-sensitive liposome consisting of DOPE/O A/Choi and 1% red cell into PBS buffers with different pH=4.0~8.0 in 37癈. After 1.5 and 2.5 hour, centrifuge 1000 g, we measure the amount of releasing haemachrome by enzyme marked instrument. At the same time, we set up control group. The amount of releasing heamachrome in pH-6 is higher than mild and weak alk alescence. And we also find that the action of liposome and cell membrane is going saturation in specify time.(2). The affection of transfective activity of sensitive-liposome in different pH conditionsWe measure the transfective activity of sensitive-liposome wrapping 125I-IL-8 though U937 cell in different pH=4~8 conditions, and we set up control. We measure the count of isotopes before transfection and after transfection, the ratio may reflect the transfective activity of sensitive-liposome wrapping 125I-IL-8. The conclusion is that the transfective activity of sensitive-liposome in pH=6 is higher than otherconditions.(3). Experiment in cell cyrotoxicity of pH-sensitive liposomeWe put galactophore cancer 543 cell and different dose sensitive liposome, general liposome into 96 well. After incubating 24 hour in 37癈, we measure the cell livability by MTT. The conclusion shows: the cell livability can be reached 86% in certainty doses.(4). The expression of cPLA2 gene and protein of sensitive-liposome wrapping ASOND in cellAfter stimulating 24 hour by using PMA , U937 cell is incubated 4 hours with sensitive-liposome wrapping ASOND of cPLA2 in 37癈. Then stimulated 24 hours by LPS, we measure the expression of cPLA2 protein with Western Blot. And we distill the RNA of cell , RT-PCR , then electrophoresis with...