Biodegradable branched polycationic polymers with varying hydrophilic spacer lengths were synthesized from different triacrylate monomers and the amine monomer 1-(2-aminoethyl)piperazine by Michael addition polymerization. The hydrophilic spacers were varied by the number of ethyleneoxy groups in the triacrylate monomer (E/M) that ranged from 0 to 14. The polymer degradation depended on the spacer length and pH; the amount of ester degraded as determined by H-1 NMR after 14 days was 43.4 +/- 2.1% (pH 5.0) and 89.7 +/- 1.3% (pH 7.4) for the polymer with 0 E/M compared to 55.7 +/- 2.6% (pH 5.0) and 98.5 +/- 1.6% (pH 7.4) for the polymer with 14 E/M. Cell viability of rat fibroblasts after exposure to polymer solutions of concentrations up to 1000 mu g/mL remained high (above 66.9 +/- 12.1% compared to below 7.6 +/- 1.1% for polyethylenimine at a concentration of 50 mu g/mL or higher) and increased with the spacer length. The polyplexes made with all the synthesized polymers showed higher transfection efficiency (4.5 +/-1.7% to 9.4 +/- 2.0%, dependent on the polymer/pDNA weight ratio) with ail enhanced green fluorescent protein reporter gene compared to naked pDNA (0.8 +/- 0.4%) as quantified by flow cytometry. This study demonstrates that hydrophilic spacers can be incorporated into polycationic polymers to reduce their cytotoxicity and enhance their degradability for nonviral gene delivery.