Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions
Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions
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DOI：
10.1002/jgm.868
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发表时间：
2006-04-01
影响因子：
3.5
通讯作者：
Yang, YY
中科院分区：
文献类型：
--
作者：
Jiang, X;Dai, H;Yang, YY
背景壳聚糖已被证明是一种无毒、高效的体外基因转移和体内基因传递的载体。最近，我们已经证明壳聚糖/DNA纳米颗粒可以在胆道内注射后介导高水平的基因表达[1]。在本研究中，我们探讨了聚乙二醇接枝壳聚糖/DNA复合体通过胆道和门静脉输注将基因转移到肝脏的可能性。方法将分子量为5 kDa的聚乙二醇接枝到壳聚糖(分子量：47 kDa，脱乙酰度：94%)上，接枝率分别为3.6%和9.6%(壳聚糖单糖单元与聚乙二醇单糖单元的摩尔百分比)。通过测定壳聚糖-g-聚乙二醇/DNA复合体的粒径变化和抗胆汁或血清攻击的琼脂糖凝胶电泳法研究其稳定性。利用荧光素酶报告基因检测了聚乙二醇接枝对人肝癌细胞系HepG2基因转染率的影响。用注射器泵通过胆管和门静脉输注将壳聚糖和壳聚糖-g-聚乙二醇/DNA复合体输送到肝脏。基因在肝脏中的表达和在其他器官中的表达分布情况。通过测定血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性随时间的变化，检测壳聚糖和壳聚糖-g-聚乙二醇/DNA复合物的急性肝毒性。结果壳聚糖和壳聚糖-g-聚乙二醇在HepG2细胞中的基因转染率相当。与血清和胆汁作用后，壳聚糖-g-聚乙二醇/DNA复合体，尤其是由壳聚糖-g-聚乙二醇(GD=9.6%)制备的复合体，没有形成像壳聚糖/DNA复合体那样的大聚集体，但在30min内保持稳定。此外，壳聚糖-g-聚乙二醇在血清和胆汁存在的情况下防止了DNA的降解。胆管注射后第3天，壳聚糖-g-聚乙二醇(GD=9.6%)/DNA复合体介导的肝脏基因表达水平是壳聚糖/DNA复合体的3倍，其他器官的基因表达水平高于壳聚糖/DNA复合体。门静脉输注后第1天，壳聚糖/DNA复合体诱导的基因表达水平几乎检测不到，但壳聚糖-g-聚乙二醇(GD=9.6%)介导了显著的转基因表达。有趣的是，门静脉输注后，壳聚糖-g-聚乙二醇/DNA复合体在其他器官中的转基因表达随着聚乙二醇接枝度的增加而增加。丙氨酸氨基转移酶和天冬氨酸氨基转移酶检测结果表明，壳聚糖接枝到壳聚糖上后，对肝脏的急性毒性降低。结论壳聚糖-g-聚乙二醇作为一种安全、稳定的肝脏基因载体具有潜在的应用前景。版权所有(C)2006 John Wiley&Sons，Ltd.
Background Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion [1]. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions.Methods PEG (Mw: 5 kDa) was grafted onto chitosan (Mw: 47 kDa, deacetylation degree: 94%) with grafting degrees of 3.6% and 9.6% (molar percentage of chitosan monosaccharide units grafted with PEG). The stability of chitosan-g-PEG/DNA complexes was studied by measuring the change in particle size and by agarose gel electrophoresis against bile or serum challenge. The influence of PEG grafting on gene transfection efficiency was evaluated in HepG2 cells using luciferase reporter gene. Chitosan and chitosan-g-PEG/DNA complexes were delivered to the liver through bile duct and portal vein infusions with a syringe pump. Gene expression in the liver and the distribution of gene expression in other organs were evaluated. The acute liver toxicity of chitosan and chitosan-g-PEG/DNA complexes was examined by measuring serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST) activities as a function of time.Results Both chitosan and chitosan-g-PEG displayed comparable gene transfection efficiency in HepG2 cells. After challenge with serum and bile, chitosan-g-PEG/DNA complexes, especially those prepared with chitosan-g-PEG (GD = 9.6%), did not form large aggregates like chitosan/DNA complexes but remained stable for up to 30 min. In addition, chitosan-g-PEG prevented the degradation of DNA in the presence of serum and bile. On day 3 after bile duct infusion, chitosan-g-PEG (GD = 9.6%)/DNA complexes mediated three times higher gene expression in the liver than chitosan/DNA complexes and yielded background levels of gene expression in other organs. On day 1 following portal vein infusion, gene expression level induced by chitosan/DNA complexes was hardly detectable but chitosan-g-PEG (GD = 9.6%) mediated significant transgene expression. Interestingly, transgene expression by chitosan-g-PEG/DNA complexes in other organs after portal vein infusion increased with increasing grafting degree of PEG. The ALT and AST assays indicated that grafting of PEG to chitosan reduced the acute liver toxicity towards the complexes.Conclusion This study demonstrated the potential of chitosan-g-PEG as a safe and more stable gene carrier to the liver. Copyright (c) 2006 John Wiley & Sons, Ltd.