细胞膜表面糖胺聚糖抑制多聚酰胺胺树枝状聚合物-pDNA 转染的核内摄取但促进核后过程。

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

机构信息

Department of Pharmaceutics and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.

出版信息

Eur J Pharm Sci. 2013 Jan 23;48(1-2):55-63. doi: 10.1016/j.ejps.2012.10.016. Epub 2012 Nov 3.

DOI:10.1016/j.ejps.2012.10.016

PMID:23131796

Abstract

BACKGROUND

Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now.

METHODS

The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay.

RESULTS

Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study.

CONCLUSION

Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.

摘要

背景

细胞表面糖胺聚糖（GAGs）与非病毒载体的相互作用似乎是改变基因复合物细胞内靶位的一个重要因素。目前为止，尚未研究多聚酰胺胺（PAMAM）树枝状大分子作为非病毒载体，在细胞摄取、核内传递和质粒 DNA 的转基因表达方面，与细胞表面 GAGs 的细胞内动力学。

方法

通过光相关光谱、原子力显微镜、Zeta 测量和琼脂糖凝胶电泳对 PAMAM-pDNA 复合物的物理化学性质进行了表征。使用各种体外细胞模型，如人胚肾细胞、中国仓鼠卵巢细胞及其缺乏细胞表面 GAGs 或硫酸乙酰肝素蛋白聚糖（HSPGs）的突变体，测定不同氮磷（N：P）比的复合物的转染效率和毒性。通过流式细胞术测定复合物的细胞摄取、核摄取和转染效率，并通过优化细胞-核分离，进行实时定量 PCR 和荧光素酶测定。

结果

物理化学研究表明，PAMAM 树枝状大分子与 pDNA 结合效率高，形成具有高正 Zeta 电位的小复合物，在 N：P 比值约为 5 及更高时可适当转染细胞。N：P 高于 10 时可能会出现细胞毒性问题。GAGs 的消除导致 pDNA 核摄取增加近一个数量级，转染效率提高 2.6 倍以上，高于 CHO 亲本细胞。然而，只有细胞表面 HSPGs 的消除并不能显著影响核摄取的 AUC 和转染的 AUC，并且本研究中报道了与 GAGs 对使用 PAMAM 作为递送载体的核内 pDNA 的影响相关的有趣数据。