Investigation On Accelerated Blood Clearance Of Pegylated Gambogenic Acid Liposomes And Its Preliminary Mechanism

Objective:(1)Prepare the preparation of ABC phenomenon model formulation PEG-GNA-Liposomes,select the best production method and prescription,and then carry out characterization analysis;(2)Prove second injection of PEG-GNA-Liposomes will produce ABC phenomenon;review the interval between two injections and influence of dosage on the performance of the phenomenon;at the same time,review the amount of the drug remained in the liver and spleen;(3)Analyze ABC phenomenon from the perspectives of hematology and histology.The aim is to find the solution for removing ABC phenomenon from the second injection of PEG nano-carrier.Methods:(1)By referring to previous researches,taking encapsulation efficiency as the main indicator,and carrying out single fact study and orthogonal test,it finalizes solvent-injection method as the best way to make PEG-GNA-Liposomes.Carry out characterization analysis of PEG-GNA-Liposomes via transmission electron microscope,particle size,Zeta potential,and release in vitro,etc.;(2)Nail down the HPLC method to test GNA in plasma,liver and spleen.Compare the speed of curve dropping in first injection to that in second injection of PEG-GNA-Liposomes in order to review whether there will be ABC phenomenon in the second injection.Set up three groups with different intervals between two injections,i.e.3 days,5 days,and 7 days,and compare the speed of curve dropping,in a bid to review its dependency on duration.Set up three groups with different amounts of dosage in order to review its dependency on dosage.In my first experiment,two injections of three groups both follow the pattern of high,medium,and low dosage.The result shows that the ABC phenomenon of the high dosage group is the strongest while the medium dosage is the weakest.Considering it might be due to different dosage in the second injection,I readjust my experiment.In the second experiment,the first injection follows the pattern of high,medium,and low dosage and second injection of three groups all adopt medium dosage,in order to review the influence of different dosage of first injection on the ABC phenomenon of second injection.At the same time,the amount of the drug remained in the liver and spleen will be tested by HPLC 30 minutes after injection;(3)Review the changes in the amount of anti-PEG-Ig M under different intervals and dosages by ELISA.Review changes in the amount of GNA raw material medicine group,and P-gp,ABCA1,and ABCG1 transporter in blank PEG-Liposomes group,and PEG-GNA-Liposomes group three days after injection.Results:(1)Nail down the best production method and prescription for making PEG-GNA-Liposomes: HSPC: 135mg;cholestenone: 11.3mg;m PEG-PE: 11.3mg;organic solvent of ethanol: 2m L;tetrahydrofuran: 1ml;and distilled water: 24 m L.The temperature will be 60? and churning time is three hours.Under the best scenario,PEG-GNA-Liposomes will be quasi-ball size with an average partical size of 124.4nm and Zeta potential value of-29.6m V.The drug remains stable within 30 days in the 4?environment.Its release in vitro accords with the Higuchi formula,accompanying with slowly releasing effect.(2)ABC phenomenon is witnessed in the second injection of PEG--GNA-Liposomes,which will become weaker as the duration extends longer.If two injections of three groups both follow the pattern of high,medium,and low dosage,the result shows that the ABC phenomenon of the high dosage group is the strongest while the medium dosage is the weakest.If the first injection follows the pattern of high,medium,and low dosage and second injection of three groups all adopt medium dosage,the ABC phenomenon weakens as the dosage of the first injection declines.(3)According to the result of Ig M testing result,the amount of anti-PEG-Ig M declines as the duration extends after first injection of PEG-GNA-Liposomes.If the injection of PEG-GNA-Liposomes follows the pattern of high,medium,and low dosage,the amount of anti-PEG-Ig M five days after injection will decline as the injection dosage declines.It is concluded that the amount of anti-PEG-Ig M relates to duration and dosage.According to WB results,an increase is witnessed in the amount of MDR1,ABCA1,and ABCG1 transporter in blank PEG-Liposomes group,and PEG-GNA-Liposomes group,while no increase is witnessed in GNA raw material medicine group.Conclusion:ABC phenomenon will be witnessed in the second injection of PEG-GNA-Liposomes,which relates to dosage and interval.The strongest ABC phenomenon will be witnessed under a dosage of 1.825mg/kg and interval of three days.The amount of anti-PEG-Ig M relates to ABC phenomenon: the more anti-PEG-Ig M,the stronger ABC phenomenon.However,as per the WB result,the result of ABC transporters is inconsistency with the amount of drug remained in the liver,which might be due to the influences of multiple metabolic pathways and demands further research and study.