Construction And Biological Evaluation In Vitro And In Vivo Of Polyethyleneimine-based Gene Delivery Vectors

Background Gene therapy is a kind of biomedical therapy by regulating the expression of specific genes,such as nucleic acids,antisense oligonucleotides or small interfering RNAs,to achieve the purpose of treating some diseases.It can be used not only to hereditary diseases,but also to the acquired diseases,such as cancer,viral infection,diabetes and AIDS.The successful gene therapy requires efficient gene delivery vectors,which can be divided into viral vectors and non-viral vectors.Due to the restriction of immunogenicity,the size of delivery gene fragments and industrial production,the clinical application of viral vectors is greatly limited.Development of non-viral vectors has become a hot field in gene therapy because of the lower production cost,cytotoxicity,immunogenicity,and unrestricted size of delivery gene fragments.Polyethyleneimine(PEI)is a kind of cationic non-viral gene delivery vectors which is widely studied.PEI can exhibit stable and efficient gene expression under different conditions,and PEI25 k is regarded as the "golden standard" for gene delivery.The high density of primary,secondary and tertiary amine groups allows PEI to have significantbuffering capacity over a wide range of p H values,which is called "proton sponge effect" and may be the key reason for its good transfection performance.However,there are still some shortcomings such as low transfection efficiency,lysosomal degradation and high cytotoxicity.Therefore,it has important theoretical and practical value for the construction of the gene delivery vectors based on PEI with high transfection activity and low toxicity.Objectives In this thesis,two novel kinds of PEI-based gene delivery vectors were designed and synthesized by using linear and branched PEI25 k as the starting materials.By using LPEI25 k and α-cyclodextrin as starting materials,pseudopolyrotaxane was obtained,and when using 2,4-dinitrobenzaldehyde as capping group,acid-sensitive-capped and non-acid-sensitive-capped polyrotaxanes were also obtained.The mannitol-modified cationic gene delivery vectors were prepared by using BPEI25 k as starting material,which could enhance caveolae-mediated endocytosis pathway and avoid the lysosomal degradation to the loaded genes,thus obtaining the delivery vectors with high transfection activity and good biocompatibility.Methods 1.The first kind of vectors was constructed based on LPEI25 k and α-cyclodextrin.Through the host-guest interaction and using 2,4-dinitrobenzaldehyde as capping group,pseudopolyrotaxane,acid-sensitive and non-acid-sensitive polyrotaxanes were obtaind.The second kind of vectors was mannitol-modified BPEI25 k,which was prepared by using BPEI25 k and mannose as starting materials.2.The DNA condensation ability was evaluated by agarose gel electrophoresis.The particle size and Zeta potential of vectors/DNA polyplexes were characterized by Delsa? Nano C Particle Analyzer.Through the fluorescence microscopy and flow cytometry,the transfection efficiencies of the constructed gene delivery vectors were evaluated.The transfection stability of optimal gene dilivery vectors in the presence of serum was also investigated.The cytotoxicity of the optimal gene vectors was evaluated by MTT assay.3.The endocytosis pathways of LPEI25k/CD and BPEI25k-man-L were studied by using three endocytosis pathway inhibitors.The endocytic uptake mechanism of BPEI25k-man-L was studied by Western-blot experiment.4.In vivo transfection experiments were carried out in the nude mice to investigate the distribution of GFP expression delivered by the optimal gene vectors through tail vein injection.Results Part one: LPEI based pseudopolyrotaxane and polyrotaxane 1.The structures of LPEI25 k based pseudopolyrotaxane and polyrotaxane derivatives were comfirmed by 1H NMR and IR spectra.2.The mean particle sizes of LPEI25k/CD,LPEI25k/CD-PA and LPEI25k/CD-PB/p DNA complexes were 384.9,175.4 and 162.6 nm respectively,of which the PDI were 0.333,0.150 and 0.198,and the Zeta potentials were 3.57,4.03 and 5.54 m V,respectively.All three materials could form regular spherical and positively charged nanoparticles with p DNA and could be used for gene delivery.Gel retardation experiments showed that the three vectors completely retarded p DNA at the weight ratio of 1: 1,whereas LPEI25k/CD could completely retarded p DNA at a lower weight ratio of 1: 2.3.The results of gene transfection showed that compared with LPEI25 k,LPEI25k/CD showed equal or even higher transfection performance.Compared with the acid-sensitive-capped LPEI25k/CD-PA,the non-acid-sensitive-capped LPEI25k/CD-PB showed fewer positive transfected cells and lower mean fluorescence intensity under three N/P weight ratios,indicating that acid-sensitive-capped polyrotaxane exhibited better transfection efficiency than non-acid-sensitive-capped polyrotaxane.The transfection efficiencies of LPEI25 k at N/P weight ratios of 10: 1,20: 1 and 50: 1 were significantly reduced in the presence of 10% serum.As for LPEI25k/CD,however,at N/P weight ratio of 10: 1,the positive transfected cells and mean fluorescence intensity(MFI)were consistent with the transfection results without serum.When the N/P weight ratio increased to 20: 1 and 50: 1,the transfection of LPEI25k/CD was less affected by serum than LPEI25 k,showing stable transfection efficiency.4.LPEI25k/CD(IC50 = 30.7 μg/m L)exhibited lower cytotoxicity than LPEI25k(IC50 = 20.0 μg/ m L),thus showing better biocompatibility and safety.5.Protection against DNase I degradation experiment showed that both LPEI25k/CD and LPEI25 k had good DNA protecting capability.The experiment of decondensation to release gene from the delivery vectors showed that p DNA could be more easily released from LPEI25k/CD/p DNA complex than LPEI25k/p DNA.6.LPEI25k/CD/p DNA complex was uptaken mainly through clathrin-mediated endocytosis.7.In vivo experiments showed that LPEI25k/CD/p DNA complex could express GFPeffectively in nude mice.The order of fluorescence intensity in organs was testis,brain,liver,lung,kidney,spleen and heart.Part two: BPEI25 k based cationic polymer 1.The structure of the four BPEI25 k based cationic polymers was identified by 1H NMR and IR spectra.The grafting degree of mannitol was 20.8,47.0,56.2 and 171.1 for BPEI25k-man-S/L/M/H by elemental analysis,respectively.2.The mean particle size,PDI and Zeta potential of the nanoparticle formed by the three cationic polymers of BPEI25k-man-S/L/M with p DNA were similar to that of the control BPEI25 k at four N/P weight ratios.The Zeta potential of BPEI25k-man-H was equal to the control,but the mean particle size was larger.The results of gel retardation showed that BPEI25k-man-S/L/M could completely retard p DNA at N/P of 1: 2,while BPEI25k-man-H could completely retard p DNA at N/P of 1: 1.3.The transfection efficiencies of the four vectors were much lower than that of BPEI25 k at N/P of 1: 2,when N/P were 1: 2,5: 1 and 10: 1,BPEI25k-man-L had the equal positive transfected cells and MFI as the control,while the positive cells of BPEI25k-man-S and BPEI25k-man-M were lower,but with the equal mean fluorescence intensity as the control.As for BPEI25k-man-H,its positive transfected cells and MFI were lower than those of control.BPEI25k-man-L showed equal transfection efficiency as that of BPEI25 k at N/P of 2: 1,5: 1 and 10: 1,and thus chosen as a preferred carrier for the next step.In the presence of 10% serum,the positive transfected cells of BPEI25 k were significantly fewer at N/P weight ratio of 2: 1,and the average fluorescence intensity remained unchanged;but for BPEI25k-man-L at N/P weight ratio of 2: 1,the transfection activity was equal as that without serum,and exhibited stable transfection efficiency.4.BPEI25k-man-L(IC50 = 31.4 μg/m L)exhibited lower toxicity compared with BPEI25k(IC50 = 15.3 μg/m L),thus showing better biocompatibility and safety.5.Both BPEI25k-man-L and BPEI25 k had good DNA protection ability and DNA released ability.6.The results of transfection in the presence of three endocytosis inhibitors showed that the endocytosis pathway of BPEI25k/p DNA complex was mainly clathrin-mediated endocytosis in HEK293 T cell,while the endocytosis pathway of BPEI25k-man-L/p DNA complex was mainly caveolae-mediated endocytosis.The results showed that the modification of BPEI25 k by mannitol had changed the endocytosis pathway of BPEI25 k.The results of Western-blot showed the mechanism was that the mannitols on the carrierformed hypertonic environment,which could induce the phosphorylation of caveolin-1 by Src tyrosine kinase,and thus stimulated the formation of the caveolae and fall off from the plasma-membrane.7.The transfection results of in vivo experiments showed that BPEI25k-man-L/p DNA complex could express GFP effectively in the nude mice.The order of fluorescence intensity in organs was brain,testis,liver,kidney,lung,heart and spleen.Conclusions Two kinds of PEI-based gene vectors were designed and synthesized.The structure of each carrier was confirmed by 1H NMR and IR spectra.Among them,LPEI25k/CD and BPEI25k-man-L exhibited better transfection performance in vitro and stable gene expression in the presence of serum,and were chosen as the optimal vectors.The endocytosis pathways of LPEI25k/CD/p DNA and BPEI25k-man-L/p DNA complex were mainly clathrin-mediated endocytosis and caveolae-mediated endocytosis,respectively.The endocytosis mechanism of BPEI25k-man-L/p DNA complex was that the mannitols on the carriercan induce the phosphorylation of caveolin-1 by Src tyrosine kinase,and thus stimulate the formation of the caveolae and its separation from the plasma-membrane.In vivo transfection experiments showed that both LPEI25k/CD/p DNA and BPEI25k-man-L/p DNA complexes could efficiently and consistently express GFP in nude mice,demonstrating further research potential as gene delivery vectors.