Lo Yu-Lun, Chou Han-Lin, Liao Zi-Xian, Huang Shih-Jer, Ke Jyun-Han, Liu Yu-Sheng, Chiu Chien-Chih, Wang Li-Fang
Department of Medicinal & Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
Nanoscale. 2015 May 14;7(18):8554-65. doi: 10.1039/c5nr01404b.
MicroRNA-128 (miR-128) is an attractive therapeutic molecule with powerful glioblastoma regulation properties. However, miR-128 lacks biological stability and leads to poor delivery efficacy in clinical applications. In our previous study, we demonstrated two effective transgene carriers, including polyethylenimine (PEI)-decorated superparamagnetic iron oxide nanoparticles (SPIONs) as well as chemically-conjugated chondroitin sulfate-PEI copolymers (CPs). In this contribution, we report optimized conditions for coating CPs onto the surfaces of SPIONs, forming CPIOs, for magneto-gene delivery systems. The optimized weight ratio of the CPs and SPIONs is 2 : 1, which resulted in the formation of a stable particle as a good transgene carrier. The hydrodynamic diameter of the CPIOs is ∼136 nm. The gel electrophoresis results demonstrate that the weight ratio of CPIO/DNA required to completely encapsulate pDNA is ≥3. The in vitro tests of CPIO/DNA were done in 293 T, CRL5802, and U87-MG cells in the presence and absence of an external magnetic field. The magnetofection efficiency of CPIO/DNA was measured in the three cell lines with or without fetal bovine serum (FBS). CPIO/DNA exhibited remarkably improved gene expression in the presence of the magnetic field and 10% FBS as compared with a gold non-viral standard, PEI/DNA, and a commercial magnetofection reagent, PolyMag/DNA. In addition, CPIO/DNA showed less cytotoxicity than PEI/DNA and PolyMag/DNA against the three cell lines. The transfection efficiency of the magnetoplex improved significantly with an assisted magnetic field. In miR-128 delivery, a microRNA plate array and fluorescence in situ hybridization were used to demonstrate that CPIO/pMIRNA-128 indeed expresses more miR-128 with the assisted magnetic field than without. In a biodistribution test, CPIO/Cy5-DNA showed higher accumulation at the tumor site where an external magnet is placed nearby.
微小RNA-128（miR-128）是一种具有强大胶质母细胞瘤调控特性的有吸引力的治疗分子。然而，miR-128缺乏生物学稳定性，导致其在临床应用中的递送效果不佳。在我们之前的研究中，我们展示了两种有效的转基因载体，包括聚乙烯亚胺（PEI）修饰的超顺磁性氧化铁纳米颗粒（SPIONs）以及化学共轭的硫酸软骨素-PEI共聚物（CPs）。在本研究中，我们报告了将CPs包被在SPIONs表面形成CPIOs用于磁基因递送系统的优化条件。CPs与SPIONs的优化重量比为2∶1，这导致形成了一种稳定的颗粒，可作为良好的转基因载体。CPIOs的流体动力学直径约为136 nm。凝胶电泳结果表明，完全包裹质粒DNA（pDNA）所需的CPIO/DNA重量比≥3。CPIO/DNA的体外试验在293 T、CRL5802和U87-MG细胞中进行，有无外部磁场均可。在有或无胎牛血清（FBS）的情况下，在这三种细胞系中测量了CPIO/DNA的磁转染效率。与金标准非病毒载体PEI/DNA和商业磁转染试剂PolyMag/DNA相比，在磁场和10% FBS存在的情况下，CPIO/DNA表现出显著改善的基因表达。此外，CPIO/DNA对这三种细胞系的细胞毒性比PEI/DNA和PolyMag/DNA小。在辅助磁场作用下，磁复合物的转染效率显著提高。在miR-128递送中，使用微小RNA板阵列和荧光原位杂交来证明，与无辅助磁场相比，在辅助磁场作用下CPIO/pMIRNA-128确实表达了更多的miR-128。在生物分布试验中，CPIO/Cy5-DNA在附近放置外部磁体的肿瘤部位显示出更高的蓄积。
