The Studies On The Synthesis And Modification Of Cationic Poly(Organophosphazenes) Derivatives As Non-viral Vectors For Gene Delivery System

Two types of cationic polyphosphazene derivatives bearing ternary amino groups or primary amino groups were synthesized respectively in order to explore the possibility of these novel cationic polymer as gene non-viral vectors.The primary amino groups of polyphosphazene derivatives were modified with urocanic acid(UA) or lactobionic acid(LA) for enhanced endosomal escape or liver targeting.The characterization of the polymers and in vitro transfection of the polymer/DNA nanoparticles were studied.The in vivo transfection of galactosylated polyphosphazene/DNA nanoparticles in mice bearing BEL-7402 tumor was investigated as well.The polyphosphazene derivatives bearing ternary amino groups included poly(DMAEA/ Boc-his-OMe)phosphazene(PDHP) and poly(imidazole/DMAEA) phophazene(PIDP) with various degrees of subsitution of imidazole group.PDHP with 13%substitution of Boc-his-OMe group and PIDP with 17%substitution of imidazole group could condense DNA into positive charged nanoparticles with 100nm particle size at the polymer/DNA ratio of 10:1(w/w).PDHP/DNA self-assemble nanoparticles(PHSNs) and PIDP/DNA self-assemble nanoparticles(PISNs) showed one fold or double fold higher in vitro transfeciton efficiency on 293T cell line at serum free medium with 2.5μg DNA/well than PEI/DNA self-assemble nanoparticles (PESNs)respectively,also higher than that of poly(di-DMAEA)phosphazene/DNA nanopartciles(PASNs)as well.The cytotoxicities of PHSNs and PISNs were much lower than that of PESNs and PASNs.Poly(2-(2-aminoethoxy)ethano)phosphazene(PAEP)/DNA nanoparticles bearing primary amino groups could achieve efficient transfection in complete medium.They have higher cell viability compared to PEI/DNA nanoparticles.The substitution of urocanic acid had influences on the physical-chemical properties and biological characterization of PAEP/DNA nanoparticles.The positive charge density and cytotoxcity of nanoparticles decreased with increasing degree substitution of urocanic acid from 7%to 25%.The buffer capacity,in vitro transfection and cell uptake efficiency of the nanoparticles enhanced after 7%substitution of urocanic acid to PAEE The transfection of PAEP with 25%substitution of urocanic acid(UA25-PAEP),however,was much lower than PAEP with 7%substitution of urocanic acid,called UA7-PAEE The green fluorenscence of UA7-PAEP/DNA nanopariticles and PAEP/DNA nanopariticles could be observed in cytosol and nuclus while that of UA25-PAEP/DNA nanopariticles was only observed in cytosol.The lower endocytosis and buffer capacity of PAEP/DNA nanoparticles resulted in less efficient transfection than UA7-PAEP/DNA nanopariticles.The transfeciton of PAEP/DNA nanopariticles,UA7-PAEP/DNA nanopariticles and UA25-PAEP/DNA nanopariticles were insignificantly decreased in the presense of a proton pump inhibtor Bafilomycin A1.UA7-PAEP/DNA nanopariticles and UA25-PAEP/DNA nanopariticles prepared in acetate pH5.7 achieved more efficient transfeciton than nanopariticles prepared in water.In a word,UA7-PAEP/DNA nanoparticles with 7% substitution of urocanic acid could exhibit higher in vitro transfeciton efficiency with significant lower cytotoxicity compared with PAEP/DNA nanoparticles.The primary amino groups of PAEP were modified with 5%lactobionic acid(LA-PAEP)with an attempt to improve the targeting efficiency to liver.LA-PAEP could condensed DNA into nanoparticles with 130nm particle size at the polymer/DNA ratio of 20:1.LA-PAEP/DNA nanoparticles showed much more mean fluoresencent intensity as well as higher expression of luciferase on BEL-7402 cells than that on Hela cells.The transfection of LA-PAEP/DNA nanoparticles on BEL-7402 cell was much higher than that of PAEP/DNA nanoparticles.In competition test,the transfection of LA-PAEP/DNA nanoparticles was highly inhibited in the present of 20 mM galactose,indicating LA-PAEP/DNA nanoparticles was mediated by the receptor-mediated endocytosis.In vivo transfection, all of the PAEP/DNA nanoparticles,LA-PAEP/DNA nanoparticles PEI/DNA nanoparticles presented more luciferase expression in tumor compared to other organs. LA-PAEP/DNA nanoparticles showd much higher luciferae expression in tumor than unmodified PAEP/DNA nanoparticles.PEI/DNA nanoparticles showed the higest luciferase expression in tumor.Summarily,nanoparticles bearing different type of amino groups showed efficient transfection with lower cytotoxicity than PEI/DNA nanoparticles.Nanoparticles bearing primary amino groups mediated more efficient transfeciton than nanoparticles bearing ternary amino groups in complete medium.LA-PAEP/DNA nanoparticles exhibited tumor targeting in vivo transfeciton,which was coincident with that in vitro transfection on BEL-7402 cell line.In a word,our study on the synthesis and modification of poly(organophosphazenes) derivatives as non-viral vectors for gene delivery system provided a promising potential for developing safe and efficient transfecant for gene delivery.