Comparative study of nanoparticle-mediated transfection in different GI epithelium co-culture models.
Comparative study of nanoparticle-mediated transfection in different GI epithelium co-culture models.
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DOI：
10.1016/j.jconrel.2012.01.041
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发表时间：
2012-05-30
期刊：
影响因子：
--
通讯作者：
Leong KW
中科院分区：
文献类型：
--
作者：
Loo Y;Grigsby CL;Yamanaka YJ;Chellappan MK;Jiang X;Mao HQ;Leong KW
口服非病毒基因传递是最有吸引力的，可以说是最具挑战性的给药途径。为了确定合适的载体，我们研究了不同类别（天然聚合物、合成聚合物和合成脂质聚合物）的DNA纳米颗粒通过三种充分表征的肠上皮细胞模型（Caco 2、Caco 2-HT 29 MTX和Caco 2-Raji）的转运。聚（磷酰胺-二丙胺）（PPA）和脂质-鱼精蛋白-DNA（LPD）纳米颗粒在这些模型中一致地显示最高水平的人胰岛素mRNA表达和荧光素酶蛋白表达，通常比背景高至少三个数量级。所有纳米颗粒都增加了紧密连接的通透性，其中PPA和PEI在第一小时内的跨上皮电阻（TEER）下降最显著，分别为（35.3 ± 8.5%）和（37.5 ± 1.5%）。TEER降低的幅度与纳米颗粒表面电荷相关，暗示与紧密连接蛋白的静电相互作用。然而，共聚焦显微镜显示，纳米颗粒主要被肠上皮细胞吸收。定量摄取和转运实验表明，即使在24小时后，内吞的量子点（QD）标记的PPA-DNA纳米颗粒仍保留在肠细胞中。在基底侧室中检测到可忽略量的量子点标记的DNA，除了Caco 2-Raji共培养物，其与Caco 2和Caco 2-HT 29 MTX培养物相比更容易内化纳米颗粒2至3倍。PEG化使转染效率降低了至少一个数量级，降低了TEER降低的幅度，并使PPA-DNA纳米颗粒的摄取减半。一个关键的发现是在基础HepG 2细胞中检测到胰岛素mRNA，这意味着一些质粒被转运穿过肠上皮层，同时保留至少部分生物活性。然而，低效率的运输表明，转胞吞单独不会产生显着的治疗效果，这种运输方式必须通过其他手段在体内增强，使非病毒口服基因传递实用。
Oral nonviral gene delivery is the most attractive and arguably the most challenging route of administration. To identify a suitable carrier, we studied the transport of different classes (natural polymer, synthetic polymer and synthetic lipid–polymer) of DNA nanoparticles through three well-characterized cellular models of intestinal epithelium (Caco2, Caco2-HT29MTX and Caco2-Raji). Poly(phosphoramidate-dipropylamine) (PPA) and Lipid-Protamine-DNA (LPD) nanoparticles consistently showed the highest level of human insulin mRNA expression and luciferase protein expression in these models, typically at least three orders of magnitude above background. All of the nanoparticles increased tight junction permeability, with PPA and PEI having the most dramatic transepithelial electrical resistance (TEER) decreases of (35.3 ± 8.5%) and (37.5 ± 1.5%) respectively in the first hour. The magnitude of TEER decrease correlated with nanoparticle surface charge, implicating electrostatic interactions with the tight junction proteins. However, confocal microscopy revealed that the nanoparticles were mostly uptaken by the enterocytes. Quantitative uptake and transport experiments showed that the endocytosed, quantum dot (QD)-labeled PPA–DNA nanoparticles remained in the intestinal cells even after 24 h. Negligible amount of quantum dot labeled DNA was detected in the basolateral chamber, with the exception of the Caco2-Raji co-cultures, which internalized nanoparticles 2 to 3 times more readily compared to Caco2 and Caco2-HT29MTX cultures. PEGylation decreased the transfection efficacy by at least an order of magnitude, lowered the magnitude of TEER decrease and halved the uptake of PPA–DNA nanoparticles. A key finding was insulin mRNA being detected in the underlying HepG2 cells, signifying that some of the plasmid was transported across the intestinal epithelial layer while retaining at least partial bioactivity. However, the inefficient transport suggests that transcytosis alone would not engender a significant therapeutic effect, and this transport modality must be augmented by other means in vivo to render nonviral oral gene delivery practical.
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