Study On Treatment Of Cerebral Glioma Of Antitumor Drugs Mediated By Transferrin Receptor Binding Peptides And Ultrasound Microbubbles

Objectives Malignant gliomas represent the largest group of brain tumors in humans.however,there has been bad clinical curative effect and short survival period for glioma,because of the existence of Blood brain barrier(BBB)and weak selectivity of traditional antitumor drugs on tumor cells.we find that ultrasound microbubbles can open BBB noninvasively and reversibly,the transferrin receptors(TfR)are highly expressed on glioma cell membranes,and binding peptide of transferrin receptors(HAIYPRH)can specificly bind to TfR on cell membrane of gliomas.In this study,we firstly combine two targeting technology,ultrasound microbubbles can open BBB and deliver drugs to the brain,binding peptide-mediated drugs can selectively act on brain glioma,in order to improve targeting therapy of brain tumor.This study,with5-fluorouracil(5-FU)and epirubicin(EPI)as tool medicines,is aimed to evaluate antitumor activity of HAIYPRH-EPI and HAIYPRH-5-FU against cerebral glioma cells in vitro and the targeting and antitumor activity ofHAIYPRH-5-FU-coated ultrasound microbubbles against cerebral glioma activity in vivo.Methods In the first part,with EPI as a tool medicine and glutamic acid as the attachment,we prepared HAIYPRH-EPI conjugate,with 5-FU as a tool medicine and 6-aminocaproic acid as the attachment,we prepared HAIYPRH-5-FU conjugate.In the second part,the aim was to evaluate antitumor activity of the conjugates against cerebral glioma cells and its relationship with Tf R.We first detected surface TfR1 expression on LN229 and U87 glioma cells membrane by flow cytometry method,then by regarding tumor cells(U87 and LN229 glioma cells)as study objects,cell proliferation inhibition was tested by MTT assay to calculate survival rates,apoptosis was observed by transmission electron microscopy(TEM)and caspase 3,8,9 activation detection kits,qualitative and semiquantitative analysis of cell uptake of HAIYPRH-EPI was done by fluorescence microscope and flow cytometry respectively,and quantitative analysis of cell uptake of HAIYPRH-5-FU was done by HPLC-MS/MS method.In the third part,we prepared HAIYPRH-5-FU-coated ultrasound microbubbles by optimal formulations from orthogonal experimental design,calculated encapsulation rate and drug loadings,observed morphology and stability,and detected particle size,particle size distribution and potential.In the fourth part,the aim was to evaluate the targeting and antitumor activity of HAIYPRH-5-FU-coatedultrasound microbubbles against cerebral glioma activity in vivo,we established rat LN229 glioblastoma model by stereotaxic implantation,verified the correctness of the model with MRI and HE staining,measured the concentration of 5-FU and HAIYPRH-5-FU in tissues and blood to investigate the targeting efficiency,observed the inhibition effect of conjugate-coated ultrasound microbubbles on glioma,and compared survival time and weight of each subgroup rats.Results In the first part,we prepared HAIYPRH-EPI conjugate and HAIYPRH-5-FU conjugate successfully.The molecular weight of HAIYPRH-EPI and HAIYPRH-5-FU detected by mass spectrometry was consistent with expectations,the purity was 99.03% and 99.07% by HPLC respectively.In the second part,the results of TfR1 expression on glioma cells membrane by flow cytometry method showed,TfR1 expression on U87 cells membrane was low and TfR1 expression on LN229 cells membrane was high.Compared with U87 glioma cells with lower surface TfR expression,HAIYPRH-EPI and HAIYPRH-5-FU presented significant cytotoxic effect in a dose-dependent manner and apoptosis effect in LN229 glioma cells with higher surface TfR expression,and 25 ? M transferrin(Tf)strengthened the effect.HAIYPRH-EPI and HAIYPRH-5-FU presented significant cell uptake in LN229 glioma cells and 25?M Tf strengthened the effect,at the same time,cell uptake,apoptosis and cytotoxic effect of EPI and 5-FU by passive transport madelittle difference between LN229 and U87 glioma cells and adding of Tf was no significant effect.In the third part,with HAIYPRH-5-FU as tool medicine,taking the score of drug embedding ratio as indexes,the optimal prescriptions were determined by orthogonal test containing four factors,which had obvious effects on drug preparation including PLGA 800 mg,span 80 1.5ml,water phase volume of first emulsifier 1ml,volume of PVA45 ml.We prepared HAIYPRH-5-FU-coated ultrasound microbubbles successfully with the optimal prescriptions,the encapsulation rate and drug loadings were(45.87±0.71)% and(0.86±0.19)% respectively by HPLC-MS/MS method;when microbubbles dissolving in deionized water,they had uniform distribution without sticky,even being kept for seven days;the mean particle size and Zeta potential were(476.3±143.2)nm and-(12.8±5.86)mV,respectively.In the fourth part,LN229 glioblastoma rat model was successfully established.5-FU ultrasound microbubbles group and HAIYPRH-5-FU ultrasound microbubbles group presented significant targeting and antitumor activity against cerebral glioma.Compared with5-FU group,tumor volume was significantly decreased in 5-FU ultrasound microbubbles group at 21 days with the same dosage.Compared with HAIYPRH-5-FU group,tumor volume was significantly decreased in HAIYPRH-5-FU ultrasound microbubbles groups including low,medium and high concentration group at both 14 days and 21 days,in a dose-depended manner.Compared with blank microbubble group,5-FUgroup and HAIYPRH-5-FU group,rats weight began to present striking difference from 28 days in HAIYPRH-5-FU ultrasound microbubbles groups including medium and high concentration group,and the concentration was the greater,the weight change was smaller.The survival period for the control group,the blank ultrasound microbubbles group,5-FU(0.5mM/kg)group,HAIYPRH-5-FU(0.5mM/kg)group,5-FU ultrasound microbubbles(0.5mM/kg)group,low concentration of HAIYPRH-5-FU ultrasound microbubbles(0.1mM/kg)group,medium concentration of HAIYPRH-5-FU ultrasound microbubbles(0.5mM/kg)group,and high concentration of HAIYPRH-5-FU ultrasound microbubbles(1mM/kg)group was 26,24,34,25,45,49,52 and 63 days,respectively.Conclusion The conjugates of HAIYPRH and antitumor drugs in covalent bonds present obvious selectivity and antitumor activity against cerebral glioma cells with higher surface TfR expression in vitro,and the conjugate-coated ultrasound microbubbles present obvious targeting and antitumor activity in rat glioblastoma model with higher surface TfR expression in vivo.Therefore,the implementation of this subject may provide new theoretical and experimental basis on developing new and promising antitumor drugs.