Construction Of Chitosan Linked Pei Non-viral Gene Delivery Carrier And Its Expression In Mesenchymal Stem Cells

Objective:In this study,chitosan was chosen as the carrier of the gene carrier,and a small molecule PEI was linked to chitosan using a covalent linking method.The various properties of CP50 were characterized in this study.And studying the transfection efficiency of CP50 in MSCs,as well as the osteogenic differentiation and migration to tumor cells of MSCs.Methods:1.Synthesis and characterization of chitosan(50kDa)-PEI(CP50):Firstly,chitosan(50kDa)-PEI(CP50)were synthesized and H1-NMR and elemental analysis were used to determine whether CP50 were synthesized successfully.The acid-base titration method was used to evaluate buffering capacity of CP50.Gel electrophoresis was used to evaluated the combination degree of CP50 and DNA with different N/P ratio.CP50 with N/P ratio=40 was measured using a laser diffraction spectrometry and TEM was used to observed morphology of CP50.2.Biosafety evaluation of CP50:Zebrafish model was used to investigate toxicity of CP50 using mortality rate and hatching rate as indictors.The cell viability and transfection efficiency of CP50 in MSCs and A549 cells were investigated by MTT assay and luciferase activity.3.Evaluation of CP50 transfection and transfection Efficiency of in MSCs:Transfection efficiency of CP50 in A549 and MSCs were evluate by luciferase activity.The mechanism of intracellular transportation of CP50 was observed by transfer experiment under confocal laser scanning microscopy.Flow cytometry was used to identify surface antigens of CP50 before and after transfection.Endocytic pathway of CP50 was determined by interfering transfection process using different endocytosis inhibitors.ALP activity detection and alizarin red staining were used to determine the results of bone differentiation.Transwell assay was used to investigated the tropism of MSCs and mscs-cxcr4 for tumor cells.Results:1.Synthesis and characterization of CP50:1H-NMR and elemental analysis showed that CP50 was successfully synthesized.In this study,the particle size and zeta potential of CP50 measured using a laser diffraction spectrometry was 111±8nm and 19.25±0.56mv.Results of gel electrophoresis show that DNA can be completely wrapped by CP50 when the ratio of N/P higher than 4:1.2.Biosafety evaluation of CP50:MTT assay and zebrafish toxicity evaluation indicate that CP50 was less toxic than PEI(25 kDa).Zebrafish is a suitable animal model for CP50.3.Evaluation of CP50 transfection and transfection Efficiency of in MSCs:CP50 has best transfection efficiency in A549 and MSCs when the N/P ratio of CP50/DNA was 40:1.The effect of serum on CP50 transfection efficiency was less than that of PEI(25kDa).CP50 can enter into nucleus after transfected 2 hours.Surface antigen of MSCs were unchanged before and after transfection and still has the biological characteristics.The results of endocytosis showed that the uptake of CP50 by MSCs was mainly dependent on the mechanism of cortical protein-mediated endocytosis(Caveloae).ALP activity assay and alizarin red staining showed that CP50 could carry TG-β1 gene transfected into MSC.TGF-β1 gene was effectively expressed and induced osteogenic differentiation in MSCs.The transfected MSC-CXCR4 cells showed higher migratory capacity than naive MSCs observed in transwell migration assay.Conclusion:The new non-viral vector synthesized using chitosan(50 kDa)and PEI(1200Da)showing feasibility in gene therapy.It has higher transfection efficiency compared with CP 100 and has good application potential and is worth study in the future.