Synthesis and Characterization of Low Molecular Weight Linear Polyethylenimines for Gene Delivery

The therapeutic application of commercially available polyethylenimines (e.g., PEI 25 kD) is marred by substantial toxicity. The benefit of using lower molecular weight polyethylenimines (<25 kD) has not been fully explored because of limited availability and high molecular heterogeneity (e.g., degree of branching) among the available few. We have synthesized a series of low molecular weight linear polyethylenimines (LPEI; 2.5-25 kD) with a single synthesis scheme to minimize molecular heterogeneity. DNA formulation with the newly synthesized linear polyethylenimines resulted in the formation of stable nanoparticles (100-150 nm) of positive zeta potential. Addition of these nanoparticles onto COS-1 and HEK 293 cell cultures led to transgene expression the efficiency and cytotoxicity of which varied with the LPEI size. The lowest molecular weight LPEI (LPEI 2.5 kD) gave the smallest level of gene expression and did not exert any cytotoxicity. The transfection activity exponentially increased with higher molecular weight LPEIs reaching maximal level with 7.5 kD LPEI and was accompanied with some cytotoxicity. The transfection activity of 7.5 kD LPEI was equal to that of the higher molecular weight LPEIs including 25 kD LPEI, but caused less cytotoxicity. To achieve high transfection efficiency without substantial increase in cytotoxicity, we cross-linked LPEI 3.6 kD with a biodegradable linkage to form a multi-block copolymer (BD3.6K) of approximately 8 kD. The multi-block copolymer, BD3.6K, expressed 20-fold higher transfection activity than that of the monomer block and produced significantly lower cytotoxicity than 25 kD PEI in vitro. Following intravenous administration, plasmid/BD3.6K complexes elicited significant gone transfer in lungs, while complexes prepared with monomer block did not yield discernable transfection activity. The transfection efficiency of the systemically administered plasmid/BD3.6K complexes was 2.5-times and 70-times higher than that of linear and branched 25 kD PEI, respectively. Transfection complexes prepared with BD3.6K exhibited better tolerability than complexes prepared with 25 kD PEIs. These results demonstrate that: (1) the lower molecular weight linear polyethylenimines (<10 kD) are more suitable for gene delivery than the commercially available higher molecular weight polyethylenimines (25 kD) and (2) the cross-linking of the non-toxic low molecular weight polyethylenimines via biodegradable linkage is a viable approach to improving PEI transfection efficiency without significantly increasing the cytotoxicity.