The Study Of Angiopep-2-EGFP On Glioma Targeting And ¹⁰B Delivery Efficiency With Boron Liposome

Background:Glioma is the most lethal type of malignant brain tumor with high degree of invasion,recurrence and mortality rate.Glioma has high antagonism to radiotherapy,and the presence of blood brain barrier(BBB)makes chemotherapy drugs could not accumulate to therapeutic concentrations at tumor site,which also increased the difficulty of glioma therapy.Therefore,it is difficult to achieve the desired cure effect with traditional radiotherapy and chemotherapy.For glioma,it is imperative to explore new treatment methods.Angiopep-2(TFFYGGSRGKRNNFKTEEY)is a hot sp ot in targeted therapy research of brain tumor in recent years,which is mainly mediated by the low density lipoprotein receptor related protein(LRP)on the blood brain barrier.Related studies have indicated that Angiopep-2 has the double targeting abilities of BBB and glioma,which provides a new way for the targeted therapy of glioma.Boron neutron capture therapy(referred to as BNCT)is a new type of brain tumor treatment.Sufficient amount of 10B in cells and ultra pure thermal neutron source are two key factors to the success of BNCT.BNCT is based on targeted boron transport and aggregation in cancer cells using boron carrier,which have a strong affinity to the cancer cells and non-toxicity to normal tissue.Only when the 10B concentration in the tumor tissue reaches to therapeutic level(20-30 μg10B/g),could thermal neutron irradiation be applied to tumor.Thermal neutrons penetrate into tumor cells and the strong nuclear reaction occurs inside the cancer cells.10B produces alpha particles with strong energy,but the range is very short,only within a cell.So BNCT could effectively kill tumor cells and maintain surrounding normal tissue.For brain tumor with high infiltration,BNCT is a very effective treatment.Objective:1.Construction of prokaryotic expression vector of fusion protein ANG-E,expression and purification of Angiopep-2-EGFP;detection of targeting ability of ANG-E on human primary astrocytes and different glioma cells.2.Analysis of 10B delivery efficiency of ANG-E modified liposome boron carriers to different glioma cells and expression variation of genes and proteins after neutron irradiation.3.Construction of brain tumor model in nude mice and analysis of 10B boron delivery in nude mice with ANG-E modified liposome.Methods:1.Primers were designed according to Angiopep-2 gene sequence,PCR was used to amplify ANG-E gene and ligated to pET-22b(+)expression vector.Angiopep-2-EGFP was expressed in Escherichia coli BL21(DE3)or Rosetta(DE3)and purified with histidine affinity chromatography column.Laser scanning confocal microscopy(LSCM)was used to detect targeting ability of ANG-E to glioma cells and human primary astrocytes;2.Diameter and Zeta potential of liposome boron carrier was measured with particle size description analyzer;ICP-AES was used to detect boron encapsulation rate in liposome and boron delivery efficiency of liposome boron carrierin vitro and in vivo.CCK-8 was used to analyze cell toxicity of Angiopep-2-EGFP modified liposome boron carrier.Real time PCR and Western blotting was used to analyze expression variation of P53 gene and protein after neutron irradiation.3.HE staining of frozen sections was used to evaluate the establishment of brain tumor model.Results:1.Fusion protein Angiopep-2-EGFP was successfully expressed and purified.2.Angiopep-2-EGFP could effectively target to glioma cells,but not to human primary astrocytes;Angiopep-2-EGFP modified liposome boron carrier has no cell toxicity;Boron delivery efficiency was improved by using Angiopep-2-EGFP modified liposome boron carriers.The maximum peak occurred at 12 h,the amount of boron in cells at different time points meet the requirement for neutron irradiation;after neutron irradiation,gene and protein expression of P53 in U87MG cells were significantly increased.3.HE staining proved that brain tumor model in nude mice was successfully established.Boron delivery in tumor was improved using ANG-E modified liposome boron carriers.Conclusion:1.ANG-E could effectively target to glioma cell lines U251,U87MG,paU87 and U87△EGFR,safe to human primary astrocytes.2.Boron delivery efficiency of ANG-E modified liposome boron carrier for different glioma cells meet the minimum requirements of BNCT.3.After neuron irradiation,expression of P53 gene and protein were significantly increased in U87MG.4.Angiopep-2-EGFP modified liposome boron carrier could delivery boron into tumor with high efficiency.