Department of Biomedical Engineering, Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan , Ann Arbor, Michigan 48019, United States.
Mol Pharm. 2013 Aug 5;10(8):3013-22. doi: 10.1021/mp400103f. Epub 2013 Jul 24.
Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane's electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.
胞质核酸酶被认为在限制基于多聚物的基因传递试剂的有效性方面发挥着重要作用。为了探究细胞膜破裂对核酸酶激活、多聚物摄取和定位时的核酸酶活性以及基因表达时的核酸酶活性的影响，我们采用了一种寡核苷酸分子信标（MB）。MB 被整合到聚合物/DNA 多聚物中，进行双色流式细胞术实验以探究 MB 切割与碘化丙啶（PI）摄取、蛋白表达和多聚物摄取的关系。此外，还通过共聚焦荧光显微镜检查多聚物和切割的 MB 的定位。采用全细胞膜片钳测量质膜电导率来探究细胞膜破裂的影响。我们观察到胞质核酸酶的差异激活，其中 B-PEI 和 G5 PAMAM 树枝状大分子（G5）具有大量的活性，jetPEI 的切割较少，而 L-PEI 的活性较低。jetPEI 和 L-PEI 表现出更高的转基因表达，与观察到的 MB 寡核苷酸切割量较低相一致。尽管核酸酶活性依赖于所使用的聚合物的选择，但与通过 PI 摄取或全细胞膜片钳测量的质膜破裂程度无关。
