Ciftci K, Levy R J
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, USA.
Int J Pharm. 2001 May 7;218(1-2):81-92. doi: 10.1016/s0378-5173(01)00623-8.
Transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. Most non-viral vectors are known to internalize in the cells by endocytosis. Therefore, low transfection efficiency of non-viral vectors may be due to intracellular degradation of input DNA in the endosomes and/or lysosomes. DNA degradation can be inhibited either by inactivating the lysosomal enzymes or obliterating endosome fusion to lysosomes using lysosomotropic agents. We report here the effects of individual lysosomotropic agents such as chloroquine, polyvinylpyrolidone (PVP) and sucrose on beta-gal expression in cultured fibroblasts COS, 293 and CHO. Cell viability was influenced by type, exposure time and concentration of lysosomotropic agents. Exposure to chloroquine at high concentration (1000 microM) or more than 4 h at any concentration (10-1000 microM) caused extensive cell death, however, cytotoxicity due to sucrose (5-500 mM) and PVP (0.01-1 mg/ml) was minimal in the cell lines tested. All the agents utilized in this study enhanced the gene expression and the transfection efficiency followed the order of sucrose>chloroquine>PVP at the concentrations used in all cell lines. Results suggest that lysosomotropic agents can enhance transfection efficiency but the degree of transgene expression may be cell- and agent-specific. Of the agents studied, sucrose appears to be an attractive agent in improving gene expression without toxic effect in the cultured fibroblasts. Thus, it can be used as an excipient in the formulation of new gene delivery systems.
将质粒DNA导入哺乳动物细胞对基因治疗构成了重大挑战。大多数非病毒载体已知通过内吞作用进入细胞。因此，非病毒载体的低转染效率可能是由于输入的DNA在内体和/或溶酶体中发生细胞内降解。DNA降解可以通过使溶酶体酶失活或使用溶酶体促渗剂消除内体与溶酶体的融合来抑制。我们在此报告了单独的溶酶体促渗剂如氯喹、聚乙烯吡咯烷酮（PVP）和蔗糖对培养的成纤维细胞COS、293和CHO中β-半乳糖苷酶表达的影响。细胞活力受溶酶体促渗剂的类型、暴露时间和浓度影响。高浓度（1000 microM）的氯喹暴露或任何浓度（10 - 1000 microM）下超过4小时的暴露会导致广泛的细胞死亡，然而，在所测试的细胞系中，蔗糖（5 - 500 mM）和PVP（0.01 - 1 mg/ml）引起的细胞毒性最小。本研究中使用的所有试剂均增强了基因表达，并且在所有细胞系中使用的浓度下，转染效率遵循蔗糖>氯喹>PVP的顺序。结果表明，溶酶体促渗剂可以提高转染效率，但转基因表达的程度可能因细胞和试剂而异。在所研究的试剂中，蔗糖似乎是一种有吸引力的试剂，可在培养的成纤维细胞中提高基因表达而无毒性作用。因此，它可以用作新型基因递送系统制剂中的辅料。
