A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy

TLDR

Poly(ethylenimine) (PEI) is a widely used cationic polymer for gene transfer with high transfection efficiency, yet its cytotoxic mechanism remains poorly understood. The study demonstrates that both branched (25 kDa) and linear (750 kDa) PEI induce rapid membrane damage and subsequent mitochondrial‑mediated apoptosis in Jurkat, HUVEC, and THLE3 cells, revealing a two‑phase toxicity pathway that informs safer gene therapy and drug delivery designs.

Abstract

Poly(ethylenimine) (PEI) is a cationic macromolecule commonly used in gene transfer/therapy protocols with high transfection efficiency both in vitro and in vivo. PEI is also cytotoxic, but the molecular basis of its cytotoxicity is poorly understood. Here, we have demonstrated that branched (25 kDa) and linear (750 kDa) PEI can both induce membrane damage and initiate apoptosis in three clinically relevant human cell lines (Jurkat T cells, umbilical vein endothelial cells, and THLE3 hepatocyte-like cells). We have defined Phase I toxicity as early necrotic-like changes (30 min) resulting from compromised membrane integrity, assessed by considerable lactate dehydrogenase release and phosphatidylserine translocation from the inner plasma membrane to the outer cell surface. Phase II cytotoxicity (24 h) was due to activation of a "mitochondrially mediated apoptotic program," resulting from PEI-induced channel formation in the outer mitochondrial membrane. This led to the release of proapoptotic cytochrome c, subsequent activation of caspase 3, and alteration in mitochondrial membrane potential as a result of caspase translocation into the mitochondria. The reported observations have important implications for the design and execution of gene therapy protocols as well for controlling intracellular distribution of drugs with cationic-based polymer-delivery systems.