Synthesis And In Vitro Study Of Stearyl Protamine As A Novel Non-Viral Gene Vector

In this thesis, stearyl protamine as a kind of novel non-viral gene vector was synthesized and investigated on the characters of its DNA complexes and the transfection efficiency. The main work includes three parts as follows:1) Three kinds of stearyl protamine (SP) named as SP1, SP2 and SP3 were synthesized of coupling protamine with stearic acid in an aquatic solution system via the active ester formed by NHS and stearic acid. The content of stearyl group (w/w %) of SP was measured by acid-base microtitration of stearic acid obtained from the degradation of SP in HCL. SP1 contains 2.5% stearyl group, 3.0% for SP2 and 5.5% for SP3. Results of DLS measurement showed that SP3 would form stable aggregates in water with around 110 nm in average diameter. The size of SP3 aggregates remained equal when the concentration of SP3 raised up, which indicated that SP3 aggregates were self-assembled and thermodynamic stable in water.2) Plasmid pGL3-control, which encoding firefly luciferase was used as DNA material in the experiments and obtained from bioengineer bacteria. The result of gel retardation assay showed that DNA could be fully compacted by SP when carrier/DNA weight ratio above 1.0. Compared with that of natural protamine sulfate, the DNA package ability of SP slightly dropped with the increasing of stearyl group. Size measurement showed that all SP/DNA complexes were smaller than 150nm in average diameter at the evaluated weight ratios. Polyplexes of SP1/DNA and SP2/DNA have the sizes similar to that of protamine sulfate/DNA but SP3/DNA polyplexes were much larger. Since SP3 would form stable self-aggregates in water. This indicated a new pattern for SP3 to compact DNA. DNase I protection assay showed that SP/DNA complexes could protect DNA from degradation by DNase I.3) H1299-human lung cancer cell was used as the subject during the MTT and in virto transfection experiments. Results showed that the cytotoxicity of SP was low at the dosage ranged from 2.5μg/ml ~ 30μg/ml. No cytotoxicity was added with the increase of stearyl...