Characterization of the human serum albumin-facilitated lipofection and examination of the effect of microtubule-interfering agents on gene expression.

摘要

The major limitation of gene delivery by cationic liposomes is low transfection efficiency. Addition of a protein to the lipofection formulation has been shown to circumvent this limitation. Because human serum albumin is highly abundant and normally found in the body, it could prevent host inflammatory and immune responses to the vector, making it a good potential ligand for the facilitated-lipofection strategy. Here, the characterization of transfection complexes prepared with human serum albumin (HSA), DMRIE-C (DC) and pCMVbeta in the pancreatic cancer cell line, Panc 1 is reported. When 0.05 to 1 mug of HSA was added to the transfection formulation, the transfection efficiency in Panc 1 cells was increased 5-7 times that of the DC plus DNA formulation. Incorporation of HSA into the lipoplex resulted in a less compact structure in solution as observed by light scattering than DC plus DNA transfection complexes.; Even though endocytosis has been suggested as the main cell internalization pathway for DNA-cationic lipid complexes, there is limited knowledge about the endocytosis mechanism for the facilitated lipofection. Pretreatment of Panc 1 cells with endocytosis inhibitors suggested a moderate involvement of clathrin and the significant involvement of actin-associated macropinocytosis in the uptake of HSA-lipoplexes.; Whether the albumin-facilitated lipofection complexes utilize microtubules for intracellular trafficking to move from the plasma membrane to the nucleus is not known. To address this question, several microtubule-interfering agents were employed. In addition to modulating microtubular structures in Panc 1 cells, increased transgene and endogenous gene expression was observed. Nocodazole activated luciferase expression from an AP-1 reporter construct. Phosphorylation of both c-Jun and ATF-2 was increased in response to nocodazole treatment in Panc 1 cells. The activation of AP-1 transcription factors in response to nocodazole treatment is most likely mediated via the JNK signaling pathway as phosphorylated JNK1 and JNK2 was observed. This work examines the potential of HSA to enhance transfection efficiency, the mechanism of endocytosis for the HSA-lipoplexes, and the mode of action for the enhancement of gene expression by microtubule-interfering agents.