Poly(4-hydroxy-l-proline ester):  Low-Temperature Polycondensation and Plasmid DNA Complexation

The cytotoxicity of a polycationic gene delivery system is critical for overall gene transfer efficency. Cells targeted for transfection must be able to support transcription and translation following gene delivery if treatment is to be successful. Cumulative cellular exposure time is one factor that mediates the cytotoxicity of polycations. Therefore, we hypothesized that hydrolytically degradable polycations, such as polyesters based on amino acids, could serve to reduce the cytotoxicity of polycationic gene delivery systems. In this paper, optimization of the low-temperature dicyclohexylcarbodiimide/(dimethylamino)pyridine (DCC/DMAP)-activated polycondensation of N -carbobenzyloxy-4-hydroxy- l -proline (CBZ-4-hydroxy- l -proline) to ultimately yield poly(4-hydroxy- l -proline ester) and the resulting polymer's interaction with plasmid DNA is reported. The optimized polycondensation of CBZ-4-hydroxy- l -proline resulted in a polymer with M w = 7880. Deprotection of the CBZ group was afforded by palladium on activated carbon-catalyzed hydrogenolysis with little cleavage of the polyester backbone. The poly(4-hydroxy- l -proline ester) was able to electrostatically complex with plasmid DNA as determined by agarose gel retardation with complete retardation occurring at the DNA:polymer ratio of 1:1 (w:w). The poly(4-hydroxy- l -proline ester) also condensed plasmid DNA into nanostructures less than 150 nm at the DNA:polymer ratio 1:3 (w:w). The in vitro cytotoxicity of the polymer was compared to that of polylysine and polyethylenimine. The minimum viability of cells incubated with poly(4-hydroxy- l -proline ester) was 85%, which is excellent when compared to the cases of polylysine (20%) and polyethylenimine (2%).