A comparative evaluation of disulfide-linked and hydrophobically-modified PEI for plasmid delivery

Non-viral gene therapy has become an important approach for treatment of hereditary and acquired diseases as a result of better understanding of molecular mechanisms involved in disease development. To design more effective gene carriers, plasmid DNA (pDNA) delivery to 293T cells was investigated by using two types of polymeric carriers; polymer constructed with disulfide (-S-S-) linkages and polymers modified with hydrophobic moieties. The base polymer used for this study was 2-kDa poly(ethylene imine) (PEI2), a relatively cell-compatible but ineffective gene carrier. The -S-S- linking was achieved via Michael addition reaction using cystamine bisacrylamide (CBA), whereas hydrophobic modification by N-acylation of PEI2 amines with palmitoyl chloride (PA). The cytotoxicity of the polymers was found to be lower than that of the 25-kDa branched PEI, but both types of modifications increased the toxicity of PEI2 to some extent. The polymers were able to form polyplexes with pDNA with variable hydrodynamic sizes (130-600 nm) and ζ-potential (3.6-20.9 mV). Based on the expression of the reporter gene Enhanced Green Fluorescent Protein (EGFP), disulfide linking significantly increased the efficiency of native PEI2, which was not effective on its own. The PA-modified PEI2 was also effective for gene delivery, but disulfide linkage of this polymer did not increase its efficiency any further. Our results showed that hydrophobic modification of 2-kDa PEI significantly improved its transfection efficiency but improvements in transfection efficiency as a result of disulfide linking was dependent on the nature of the polymeric building blocks.