Targeted CRM197-PEG-PEI/siRNA Complexes for Therapeutic RNAi in Glioblastoma.
Targeted CRM197-PEG-PEI/siRNA Complexes for Therapeutic RNAi in Glioblastoma.
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DOI：
10.3390/ph4121591
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发表时间：
2011-12-16
期刊：
影响因子：
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通讯作者：
Aigner A
中科院分区：
文献类型：
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作者：
Höbel S;Appeldoorn CC;Gaillard PJ;Aigner A
RNA干扰（RNAi）允许特异性敲除肿瘤相关基因。为了诱导RNAi，小干扰rna （sirna）的递送至关重要。这对它们在体内的治疗应用尤其具有挑战性。低分子量支链聚乙烯亚胺（PEI）对包括小RNA分子在内的核酸递送是安全有效的，这是基于其静电复合siRNA分子的能力，从而保护它们免受核酸酶降解。纳米级PEI/siRNA复合物在细胞内复合物从溶酶体释放和siRNA从复合物的细胞质释放之前被细胞内吞。这些复合物的化学修饰和配体修饰旨在引入靶组织特异性，并进一步提高pei介导的siRNA递送的功效。CRM197是一种突变的、无毒的白喉毒素（DT），它结合在胶质母细胞瘤细胞中高度表达的hb - egf样生长因子/白喉毒素受体的膜结合前体。同样，生长因子多营养因子（PTN/HB-GAM/HARP）在胶质母细胞瘤中过表达，并且对肿瘤生长具有限制性，因此代表了治疗性敲低方法的一个有吸引力的靶基因。PEI进行聚乙二醇化以减少表面电荷，并通过CRM197偶联制备了修饰的PEI，用于将siRNA递送到胶质母细胞瘤细胞中。分析了新型PEI缀合物的络合效率和最佳混合比例，并对配合物的稳定性、尺寸和zeta电位进行了物理化学表征。通过敲低报告基因在细胞培养中证实了复合物的生物活性。为了治疗裸鼠皮下人类胶质瘤异种移植物，我们全身注射靶向PTN的修饰PEI/siRNA复合物。基于PTN敲低的抗肿瘤作用表明肿瘤靶向的CRM197-PEG-PEI/siRNA优于非靶向的PEG-PEI复合物。因此，我们在体内建立了靶向的基于CRM197-PEG-PEI的siRNA递送复合物，并显示了CRM197-PEG-PEI/siRNA介导的PTN敲低的治疗效果。
RNA interference (RNAi) allows the specific knockdown of tumor relevant genes. To induce RNAi, the delivery of small interfering RNAs (siRNAs) is of crucial importance. This is particularly challenging for their therapeutic applications in vivo. Low molecular weight branched polyethylenimine (PEI) is safe and efficient for nucleic acid delivery including small RNA molecules, based on its ability to electrostatically complex siRNA molecules, thereby protecting them from nuclease degradation. The nanoscale PEI/siRNA complexes are endocytosed by cells prior to intracellular complex release from the lysosome and cytoplasmic release of the siRNAs from the complexes. Chemical modification and ligand decoration of the complexes aim at introducing target tissue specificity and further increased efficacy of PEI-mediated siRNA delivery. CRM197 is a mutated, non-toxic diphtheria toxin (DT) that binds to the membrane-bound precursor of HB-EGF-like growth factor/diphtheria toxin receptor highly expressed in glioblastoma cells. Likewise, the growth factor pleiotrophin (PTN/HB-GAM/HARP) is overexpressed in glioblastoma and is rate limiting for tumor growth, thus representing an attractive target gene for therapeutic knockdown approaches. PEGylation of PEI was performed to reduce the surface charge, and by CRM197 coupling we prepared a modified PEI for siRNA delivery into glioblastoma cells. The novel PEI conjugates were analyzed for their complexation efficiency and optimal mixing ratios, and complexes were physicochemically characterized regarding stability, size and zeta potential. The biological activity of the complexes was confirmed in cell culture by reporter gene knockdown. For the therapeutic treatment of subcutaneous human gliobastoma xenografts in athymic nude mice, we systemically injected the modified PEI/siRNA complexes targeting PTN. Antitumor effects based on PTN knockdown demonstrated the advantage of tumor-targeted CRM197-PEG-PEI/siRNA over untargeted PEG-PEI polyplexes. Thus, we establish targeted CRM197-PEG-PEI-based complexes for siRNA delivery in vivo, and show therapeutic effects of CRM197-PEG-PEI/siRNA-mediated knockdown of PTN.