Fluorescent Nanoparticle Delivered dsRNA Toward Genetic Control of Insect Pests
Fluorescent Nanoparticle Delivered dsRNA Toward Genetic Control of Insect Pests
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荧光纳米颗粒传递双链RNA以实现害虫的遗传控制
DOI：
10.1002/adma.201301201
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发表时间：
2013-09-06
影响因子：
29.4
通讯作者：
Shen, Jie
中科院分区：
文献类型：
--
作者：
He, Bicheng;Chu, Yuan;Shen, Jie
近年来，利用非病毒载体传递基因或药物因其更高的安全性和易于大规模生产而受到越来越多的关注。用于非病毒基因传递的最常用材料包括阳离子聚合物、树枝状大分子以及功能化纳米颗粒。[1]阳离子聚合物具有明确的分子结构、精确控制的化学结构和官能团，被认为是很有前途的非病毒基因传递载体。阳离子聚合物没有病毒载体引起的炎症、免疫原性和致突变作用的风险。前者能够通过静电相互作用将核酸结合和凝聚成稳定的复合体，从而保护基因不被降解，促进细胞摄取。[2]一般来说，聚合物/基因复合体的细胞毒性、包埋和降解是限制基因有效传递到细胞质中的关键障碍。[3]因此，基因治疗的临床应用需要高效率和低细胞毒性的载体。RNA干扰(RNAi)是一种特定基因的转录后沉默，可由双链RNA(DsRNA)通过靶基因内源mRNA的序列特异性降解而诱导。DsRNA介导的RNAi已成为研究基因功能的最有前途的工具之一，在基因治疗和害虫的遗传控制方面显示出巨大的应用潜力。[4]到目前为止，已实现了三种将dsRNA导入昆虫的方法，包括显微注射、口服饲喂和转基因表达。然而，仅通过口服dsRNA在幼虫细胞中抑制基因表达的效率很低，尤其是在鳞翅目昆虫中。这可能是由于分隔食物和肠道细胞的围食膜的限制，从而导致细胞摄取dsRNA的效率较低。因此，有必要探索更有效的方法来确保基因敲除。利用非病毒基因递送系统的优势，纳米基因载体将理想地促进RNAi口服喂养的应用。然而，到目前为止，基因载体介导的RNAi用于口服的方法还没有在动物体内应用的探索。在这里，我们报道了一种阳离子核壳荧光纳米颗粒(FNP)，它可以快速地被吞噬到活细胞中，具有低细胞毒性和高转染率。通过将dsRNA/FNP复合体混合到昆虫的饲料中，我们成功地利用FNP作为一种有效的基因载体来下调关键发育基因的表达，并杀死昆虫。通过荧光检测，可以对所有相关过程进行监控。这是第一个将荧光纳米颗粒传递的RNAi用于消灭害虫的例子。FNP由位于中心的荧光-3，4，9，10-四羧基二亚胺发色团(PDI)和两种不同类型的外壳组成(方案1)。中心PDI发色团是一种受欢迎的染料和颜料，因为它具有良好的化学、热稳定性和光化学稳定性，可以通过荧光显微镜检测细胞膜的运输。[6]FNP溶液在自然光下暴露2天后，荧光强度没有变化。内层由刚性的聚苯撑树枝状大分子组成，能够防止PDI发色团在水中聚集[7]，外层由柔性阳离子聚合物外壳形成。最外面的聚合物外壳是…
In recent years, the use of non-viral vectors to deliver genes or drugs has become more and more attractive due to their higher safety and ease of mass production. The most common materials used for non-viral gene delivery include cationic polymers and dendrimers as well as functionalized nanoparticles.[1] Cationic polymers with well-defined molecular architectures, precisely controlled chemical structures and functional groups, are considered as promising non-viral vectors for gene delivery. Cationic polymers lack the risk of inflammatory, immunogenic, and mutagenic effects caused by viral vectors. The former are able to bind and condense nucleic acids into stable complexes through electrostatic interaction, which can protect the gene from degradation and facilitates cell uptake.[2] In general, the cytotoxicity, entrapment and degradation of the polymer/gene complexes within endolysosomal compartments are the key barriers that restrict the efficient delivery of genes into cytoplasm.[3] The vectors with high transfection efficacy and low cytotoxicity would therefore be required for the clinical application of gene therapy. RNA interference (RNAi) is the post-transcriptional silencing of a specific gene which can be induced by doublestranded RNA (dsRNA) through sequence-specific degradation of the target endogenous mRNA. dsRNA-mediated RNAi has emerged as one of the most promising tools to study gene function and exhibited tremendous application potential for gene therapy and the genetic control of insect pests.[4] So far, three types of methods have been achieved to deliver dsRNA into insects including microinjection, oral feeding, and transgenic expression.[5] Among them, oral feeding would be the simplest way. However, the repression efficiency of gene expression is low by oral feeding of dsRNA alone in larval cells, particularly in lepidopteran insects. This is probably due to the restriction of the peritrophic membrane which separates the food and gut cells and thus leads to low efficiency of cellular uptake of dsRNA. Therefore, it is necessary to explore more efficient methods to ensure the gene knockdown. Taking the advantage of non-viral gene delivery systems, nanoparticle gene carriers would ideally facilitate the application of oral feeding of RNAi. However, up to now, gene carrier mediated RNAi for oral feeding methods has not been explored in vivo application of animals. Herein, we report a cationic core-shell fluorescent nanoparticle (FNP), which can be rapidly engulfed into live cells with low cytotoxicity as well as high transfection efficacy. By mixing the complex of dsRNA/FNP into the insect’s diet, we have successfully utilized the FNP as an efficient gene carrier to knock down key developmental gene expression and kill insect pests. By means of fluorescence detection all relevant processes could be monitored. This is the first example of fluorescent nanoparticle delivered RNAi for the elimination of insect pests. The FNP consists of a fluorescent perylene-3, 4, 9, 10-tetracarboxydiimide chromophore (PDI) in the center and two different types of outer shells (Scheme 1). The central PDI chromophore is a popular dye and pigment due to its excellent chemical, thermal, and photochemical stability, which allows the detection of the cell membrane transport via fluorescence microscopy.[6] No changes in the fluorescence intensity of the FNP solution were found after 2 days’ exposure under natural light. The inner layer is composed of a rigid polyphenylene dendrimer and capable of preventing the PDI chromophore from aggregation in water,[7] while the outer layer is formed by flexible cationic polymer shells. The outmost polymer shells …