Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate

Abstract

One of the most important requirements for non-viral gene delivery systems is the ability to mediate high levels of gene expression with low toxicity. After the DNA/vector complexes are taken up by cells through endocytosis, DNA is typically contained within the endocytic compartments and rapidly degraded due to the low pH and hydrolytic enzymes within endosomes and lysosomes, limiting its accessibility to the cytosol and ultimately to the nucleus. In this study, the endosomolytic protein listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes was conjugated with polyethylenimine (PEI) of average molecular weight 25 kDa (PEI25) via a reversible disulfide bond (LLO-s-s-PEI), and incorporated into plasmid DNA condensed with disulfide-crosslinked low molecular weight PEI 1.8 kDa (PEI1.8).We have investigated and demonstrated that high gene transfection efficiency, which is comparable to that by the most commonly used PEI25, can be achieved by reversibly crosslinking low molecular weight PEI (PEI1.8) using disulfide bonds, with greatly reduced cytotoxicity of the PEI. The reversible incorporation of LLO into the DNA condensates of PEI, through the use of the synthesized LLO-s-s-PEI conjugate, further enhances the transfection efficiency beyond that of DNA condensates with disulfide-crosslinked PEI1.8 alone.

Disulfide‐Containing Hyperbranched Polyethylenimine Derivatives via Click Chemistry for Nonviral Gene Delivery

“…[8] Low-molecular-weight (LMW) PEIs such as 1.8-kDa PEI and 0.8-kDa PEI are reported to have much lower cytotoxicity with poor gene transfection efficiency. [8,9] In order to reduce the cytotoxicity and enhance carrier unpacking into the cytosol and/or nucleus, biodegradable cationic polymers have been designed with either hydrolytically degradable ester groups [6,10] or reducibly degradable disulfide linkages [11][12][13][14] for gene delivery systems. Recently, Göpferich et al [15] have published an excellent review on the reducible disulfide-containing polymeric carriers for delivery of nucleic acids.…”

Bioreducible Polycations as Shuttles for Therapeutic Nucleic Acid and Protein Transfection

“…In some cases, the reduction of disulfide bonds facilitates this delivery process. Such reduction-activated cytotoxins have been utilized in drug delivery; for example, listeriolysin-SS-oligocation conjugates for pDNA delivery (11,90) or numerous immunotoxins (24,25,101). Commonly used synthetic protein delivery systems include cationic lipids (13,14,62), protein transduction domains (45), such as natural penetratin (85,93) and HIV-TAT (45,103), or artificial oligoarginines (93) and cationic polymers (17,30,59).…”

Synthesis of aminated polysorbate 80 for polyplex‐mediated gene transfection

“…However, viral vectors can pose certain disadvantages in that they are difficult to produce on a large scale and can potentially trigger oncogenic effects and stimulate an adverse immune response in clinical applications. [1][2][3][4][5][6][7][12][13][14][15] Nonviral synthetic vectors, although less effective in gene delivery relative to viral vectors, have a higher gene loading capacity relative to those of the viral carriers. In addition, synthetic vectors potentiate their future clinical application due to their ease of preparation and their reduced effects on the immune response.…”

“…In addition, synthetic vectors potentiate their future clinical application due to their ease of preparation and their reduced effects on the immune response. 1,[4][5][6][7]14,16 In general, cationic lipids (lipoplexes) and polymers (polyplexes) are the two major types of nonviral vectors used in gene delivery. The addition of amine groups to these vectors introduces important cationic properties to the complex that can initiate condensation and complexation with the negatively charged DNA of the gene through electrostatic interactions.…”