**3.2 Polyethylenimine derivatives**

Low molecular weight PEI (below 2 kDa) normally displays lower cytotoxicity, but inferior transfection capability as compared to high molecular weight counterparts. Klibanov *et al*. modified the primary amines of 2k-Da PEI with dodecyl or hexadecyl iodides (M. Thomas & Klibanov, 2002). The transfection efficiencies of these alkylated 2k-Da PEI are surprising. In the transfection towards COS-7 cells, dodecylated or hexadecylated 2k-Da PEI can induce a high level of gene expression in the presence of serum, that is, 5-fold higher than that of 25k-Da PEI. The cytotoxicity of these alkylated PEI is much lower as compared to 25k-Da PEI (100% *vs*. 80% cell viability).

The incorporation of poly(ethylene glycol) (PEG) into PEI may yield PEGylated PEI with reduced cytotoxicity (C.-H. Ahn et al., 2002). PEGylated PEI copolymers can be synthesized by coupling activated PEG (2000 Da) with low molecule weight PEI (600, 1200 or 1800 Da). An optimal PEI-PEG copolymer is found that has 87 units of PEI1800 and 100 units of PEG2000. Again, the copolymer can efficiently bind plasmid DNA to form nanoscale polyplexes with positively surface charge (+20~+40mV) (average diameter 120~150nm) at N/P ratios from 1/1 to 4/1. The transfection efficiency of these polyplexes towards 293T cell is 3-fold higher than that of parent PEI1800. The cytotoxicity is very low with 80% cell viability. It should be noted that PEGylation often leads to reduced transfection efficiency *in vitro*. This is because PEGylated PEI-based polyplexes have low surface charges which impair efficient cellular internalization and also efficient endosomal escape of polyplexes (Mishra et al., 2004). Thus, the molecular weight of PEG and the composition ratio between PEI and PEG must be optimized.
