Research About Construction Of Sustained-release Nanoparticles Of MPEG-CS-cRGD/Bmi-1RNAi-PTX And Its Killing Effect On Laryngeal Carcinoma Cells In Vitro

Purpose:The research is to build mPEG-CS-cRGD/Bmi-1RNAi-PTX nano sustained-release particles,and to study their features about physics and chemistry such as morphology and particle size.Meddle Hep-2 cells in vitro by using it,and its killing effect on laryngeal cancer cells can be found,which will lay a foundation for clinical treatment of laryngeal cancer biotherapy.Method:1.Three sets of Bmi-1 shRNA and one NC sequence are designed and connected with PCO24-pGensil-1 plasmid which aim to construct three sets of Bmi-1RNAi recombinant plasmids and one set of invalid recombinant plasmids.Gene sequencing is then performed.2.The expression of Bmi-1 gene in 293T cells is detected by RT-qPCR,which are transfected by recombinant plasmid.Then the interference plasmid with the best effect is selected.3.mPEG-CS nanoparticles are prepared by dialysis method,characterized by ~1H—NMR.DNA protection,gene entrapment ability are investigated by Agarose Gel electrophoresis,and cell poisonousness is checked by MTT method.4.mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticle is composed by the method of ultrasonic vibration and simple complexation.The morphology,particle size and zeta potential are examined,packet encapsulation efficiency and drug loadings are calculated.5.MTT experiment is divided into 4 groups:PTX,mPEG-CS/PTX,mPEG-CS-cRGD/PTX and mPEG-CS-cRGD/Bmi-1RNAi-PTX groups.Investigated the effect of nano preparations on the activity and proliferation of Hep-2 cells for 24h and 48h in each group,and investigated the killing effect of the nano preparations on laryngeal cancer cells.Results:1.The results of gene sequence shows that Bmi-1shRNA is successfully inserted into PCO24-pGenesil-1 plasmid carrier.RT-qPCR confirmed that all three interfering plasmids can effectively hold up the expression of Bmi-1 gene.The relative expressions of Bmi-1 are 0.118±0.003,0.124±0.017,0.172±0.007.2.The results of~1H-NMR shows that CS is successfully linked with mPEG to form the nano-carrier of mPEG-CS.MTT experimental results shows that the cytotoxicity of mPEG-CS vector does not increase with the increase of carrier concentration,the results of Agarose Gel electrophoresis shows that the gene is completely encapsulated by mPEG-CS when the ratio of w/w is 10:1,and the gene is completely protected when the ratio of w/w is24:1.3.The perfect material mixture ratios are mPEG-CS-cRGD/Bmi-1RNAi-PTX globular,partical size:182±30.6nm;zeta potential:10.14±0.52mV;entrapment rate:84.84%;drug loading:4.24%.MTT experiment shows that mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles has the strongeat killing power.And it depends on the time and consistence.Conclusion:Bmi-1RNAi,nano-carrier of mPEG-CS and mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles are successfully constructed.The interference effect of Bmi-1RNAi plasmid is obvious,and the nano vector of mPEG-CS is well biocompatible and safe.mPEG-CS-cRGD/Bmi-1RNAi-PTX nano sustained-release particles have obvious killing effect on laryngeal cancer Hep-2 cells which are ideal targeted drugs.