The Research On Cediranib And Paclitaxel-loaded Bilirubin Nanosystem For Target Therapy Of Glioma

Glioma is one of the most difficult malignant tumor to cure,and the blood-brain barrier(BBB),which exists in glioma,greatly limits the efficiency of the nano-delivery drug system in delivering chemotherapy drugs to glioma.Transferrin receptor(TfR)is highly expressed on the vascular side of cerebral capillary endothelial cells and on the surface of glioma cells,which makes it a good target for targeting glioma through BBB.D-T7(sequence for haiyprhc)is a D-configuration polypeptide with high specific affinity to TfR,and avoids hydrolysis of protease and shows high stability in vivo,which makes it a good target molecule for glioma.D-T7 polypeptide modified nanocarriers can specifically bind TfR on BBB,which promotes the nano-delivery drug system modified by D-T7 polypeptide to pass through BBB and accumulate in glioma by binding to the high expression of TfR in brain tumor cells,to achieve target drug delivery for glioma.Glioma is a complex system with low pH,a large number of reactive oxygen species(ROS)and more neovascularization,and PEGylated bilirubin nanoparticles(BRNPs)show anti-inflammatory and ROS responsiveness,and bilirubin is a natural source material with good safety,so BRNPs was chosen as the carrier system in this study.The combination of cytotoxic drugs and anti-neovascularization drugs can effectively improve the anti-glioma effect,therefore,paclitaxel(PTX,chemotherapy drug)and cediranib(CD,anti-neovascularization drug)were selected as model drugs in this study,and drugs were encapsulated in BRNPs(CD&PTX@BRNPs),and the DT7 polypeptide was further modified to the surface to obtain a target drug delivery system for glioma(CD&PTX@BRNPs),to achieve the best glioma target drug delivery and anti-glioma effect.In the second chapter,the proportion of drug combination was determined.Firstly,by investigating the effects of CD and PTX on the viability of HUVE cells,the 50% inhibitory concentration of CD and PTX on HUVE cells was 5.6 ?M and 0.25 ?M,respectively.Then,based on this,different proportions of the two drugs were designed to investigate their synergistic inhibitory effect on HUVE cells.The results showed that when PTX was 0.025 ?M and CD was 0.10 ?M,the corresponding combination index(CI)value was between 0.1 and 0.2,and when HUVE cells were incubated at a series concentration of CD/PTX of 4/1,the CI value was mostly below 1,suggesting that the combination of this proportion of drugs obtained synergistic effect.In the third chapter,bilirubin nanoparticles were constructed.Uniform size bilirubin nanoparticles were synthesized by emulsifying solvent evaporation method.The particle size,potential and PDI of CD@TBRNPs and PTX@TBRNPs nanoparticles were 112.3 nm,11.33 mV,0.346 and 117.8 nm,12.2 mV,0.336,respectively.Then the contents of CD and PTX in CD&PTX@TBRNPs were determined by HPLC,and the drug loading capacity and encapsulation efficiency of CD@TBRNPs and PTX@TBRNPs were 8.80%,98.94% and 8.51%,94.56%,respectively.In the fourth chapter,the cellular level of bilirubin nanoparticles in vitro was evaluated.Firstly,the uptake efficiency of Cou6@TBRNPs by C6 cells,bEnd.3 cells and co-culture model were qualitatively investigated by laser confocal microscopy,furthermore,the uptake efficiency of Cou6@TBRNPs by two kinds of cells was quantitatively investigated by flow cytometry.The results showed that Cou6@TBRNPs obtained strong fluorescence intensity on both cells,and the fluorescence intensity of C6 and bEnd.3 cells was 2.73 and 1.15 times higher than that of Cou6@BRNPs group,respectively.Then,cytotoxicity and apoptosis experiments were used to evaluate the toxic effects of different bilirubin nanoparticles on HUVE(simulated neovascularization in tumors)and C6 cells.The results showed that CD&PTX@BRNPs significantly inhibited the proliferation of two kinds of cells compared with one drug alone,and CD&PTX@TBRNPs more effectively inhibited the proliferation of C6 cells than CD&PTX@BRNPs group.These results indicated that the combination of the two drugs could inhibit cell proliferation and promote cell apoptosis,moreover,D-T7 polypeptide could bind with TfR on bEnd.3 and C6 cells to enhance the uptake ability of bilirubin nanoparticles on cells and the ability to penetrating bEnd.3 monolayer cells,and to enhance the effect of bilirubin nanoparticles on inhibiting C6 cell proliferation and promoting C6 cell apoptosis.In the fifth chapter,the in vivo target effect,biocompatibility and anti-glioma effect of bilirubin nanoparticles were evaluated.Firstly,the fluorescence distribution of DiD/DiR@TBRNPs in the brain of C6-bearing glioma mice was evaluated by small animal vivo imaging instrument.The results showed that the fluorescence distribution of DiD/DiR@TBRNPs in the brain of C6-bearing glioma mice was higher than that of DiD/DiR@BRNPs group,indicating that bilirubin nanoparticles modified by D-T7 peptide could pass through BBB and accumulate in glioma.After injecting CD&PTX@TBRNPs into mice,the changes of blood cell parameters and blood biochemical parameters were investigated,and the results showed that most of the parameters were within the normal range,indicating that CD&PTX@TBRNPs obtained good biocompatibility in mice and could be used to treat tumor-bearing mice.Finally,the therapeutic effects of different bilirubin nanoparticles on C6-bearing glioma mice were investigated,and the results showed that the survival time of mice in CD&PTX@TBRNPs group was 53 days,which was 3.31 times that of saline group,indicating that CD&PTX@TBRNPs significantly prolonged the survival time of tumorbearing mice.In summary,this study successfully constructed a nano-drug delivery system that can break through BBB and target glioma,and effectively improve the glioma delivery efficiency of CD and PTX,and achieve good therapeutic effect of glioma,providing a new idea for target drug delivery and treatment of glioma based on tumor microenvironment.