Research And Preparation Of Transferrin Receptor Monoclonal Antibody Tagged Baicalin Long-circulating Immune Liposomes

The preparation technology of baicalin liposome were studied by the combination of single factor analysis with orthogonal experiment.Select the lipid ratio(cholesterol and soybean phospholipid ratio),drug and the carrier ratio(soybean lecithin and baicalin ratio),hydration time,sonic disruption time.Made the entrapment efficiency and the particle size of the baicalin liposomes as the evaluating index.According to the experimental feasibility,the best prescription is dispersing membrane method,in which balcalin dissolve in methanol and then have a rotary evaporation with forming membrane solvent and the lipid ratio is 1:2,drug and the carrier ratio is 1:4,hydration time is 15 min,sonic disruption time is 15 min.the method for determing entrapment efficiency was centrifugating micro column filled by Sephadex G50 gel.Which is simple ?quickly,economic and feasibility.The particle size and zeta potential of baicalin long circulating immune liposomes was determinated,and the results meet the requirements.Here is the particle size range of three batches baicalin long-circulating liposome: The particle diameter of batch 20150527 was 126 nm + 0.12,average PDI was 0.286;particle diameter of batch 20150603 was 118 nm + 0.16 and average PDI was 0.279;particle diameter of batch 20150530 was 139 nm+ 0.09,and the average PDI was 0.267.The zeta potential range was-15 mv.-20 mv.The peak area of baicalin liposomes centrifugation liquid was determined by HPLC,then draw the curve.The peak of liposomes and free baicalin were separated obviously.The average entrapment efficiency was 27.5%.The liposomes' s morphology was observed by scanning electron microscope,and fluorescence microscopy was used to observe the fluorescence of monoclonal antibody targeted to liposome.MTT test results reflect that:(1)no matter ordinary liposome groups,long-circulating liposomes groups,immune liposomes groups,have cytotoxicity being positive correlation of time.(2)The form of liposomes make baicalin release from liposomes slowly;(3)the immune liposomes,modified transferrin receptor monoclonal antibody,can effectively combine cells.which has better cytotoxicity than long-circulating liposome.About the confirmation of Baicalin long-circulating immune liposomes targeting effect: the first is the combination(qualitative analysis): Use fluorescence microscopy to compare after medicating 60 min,Here is the fluorescent results : There is clear visible fluorescence in antibody group,and there is no fluorescent light in long-circulating liposomes group.Baicalin long-circulating immune liposome group shows a large number of clear fluorescence,Which illustrate that,baicalin long-circulating immune liposome could combine with human hepatoma cells then send out a large number of distinctive fluorescence.The second repect is a positive combination rate(quantitative analysis): the positive fluorescence rate of groups added different preparations,after the same 60 min culture time,was detected by flow cytometry.The first fluorescence positive rate of baicalin long –circulating immune liposomes is 51%;the second fluorescence positive rate of 42%,indicating that baicalin long-circulating immune liposomes and HepG2 cells indeed combinated effectivly.