PDF(Pubmed)
Abstract：
Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..
摘要：
卵巢癌是世界上最致命的妇科癌症之一。近年来,基于核酸（NA）的制剂已被证明是卵巢癌的有希望的治疗方法，包括肿瘤结节。然而,由于NAs不利的理化性质，基因治疗在临床现实中并没有那么先进，如高分子量，细胞摄取差，核酸酶快速降解，等。用于克服这些缺点的策略之一是通过与阳离子聚合物的静电相互作用来络合阴离子NA。导致所谓的聚合复合物的形成。在这项工作中,研究了pDNA和siRNA聚合复合物的大小对其渗入卵巢癌肿瘤球体的作用.为此，合成甲氧基聚(乙二醇)聚(甲基丙烯酸2-(二甲基氨基)乙酯)(mPEG-pDMAEMA)二嵌段共聚物作为与质粒DNA(pDNA)或短干扰RNA(siRNA)结合和缩合的聚合物载体。当在HEPES缓冲液（10mM，pH7.4）在5的氮/磷酸（N/P）电荷比下，形成尺寸为162±11nm的pDNA聚合复合物，而基于siRNA的聚合复合物显示25±2nm的大小。聚合复合物在相同缓冲液中具有+7-8mV的稍正ζ电位。siRNA和pDNA复合物在体外被追踪到肿瘤球体中，类似于体内无血管卵巢肿瘤结节。为此,通过将卵巢癌细胞与原代小鼠胚胎成纤维细胞以不同比例（5：2，1：1和2：5）共培养，获得了可重复的球体。渗透研究表明，在孵育24小时后，与主要位于边缘的pDNA复合物相比，siRNA复合物能够更深地渗透到同质球体（仅由癌细胞组成）和异质球体（与成纤维细胞共培养的癌细胞）中。当增加球状体中存在的成纤维细胞的比例时,聚合复合物的渗透减慢。此外，在存在血清siRNA的情况下，编码荧光素酶的多聚复合物在2D细胞中显示出高的细胞摄取，导致荧光素酶表达的约50％沉默。一起来看,这些发现表明，自组装的小siRNA复合物作为测试卵巢肿瘤结节穿透的平台具有良好的潜力。.
