Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver
Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver
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DOI：
10.1016/j.jconrel.2005.09.043
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发表时间：
2005-12-05
影响因子：
10.8
通讯作者：
Kataoka, K
中科院分区：
文献类型：
--
作者：
Miyata, K;Kakizawa, Y;Kataoka, K
用硫代化的聚乙二醇聚L赖氨酸嵌段共聚物与质粒DNA形成多离子络合物，制备了一种稳定的由聚乙二醇壳核壳型聚合物组成的冻干制剂。通过用二硫键使核心交联，避免了使用溶质保护剂。交联型聚合物胶束(CPM)在冷冻干燥和复配过程中表现出良好的稳定性，这与非交联型聚合物胶束(NCPM)在类似过程中形成的可见团聚形成了鲜明对比。在冷冻干燥/重建循环期间，需要高于13%的赖氨酸残基的硫代化度才能达到足够的CPM耐受性。动态光散射和原子力显微镜观察表明，即使在冷冻干燥后，硫代化度大于13%的CPM仍保持原有的尺寸和形状。此外，从冻干状态重构的CPM获得了与原始样品一样高的转染率。将硫代化度为37%的CPM通过眶静脉注射到小鼠体内，导致肝脏中黄色荧光蛋白变体(Venus)的基因表达非常均匀，而在游离PDNA注射的情况下，没有观察到基因表达。多链胶束核心的二硫键交联过程允许将非病毒基因载体制备为粉末制剂，而不使用任何裂解保护剂。这一成果对制药应用肯定是有用的，并显示出许多优点，包括易于调整剂量样品的浓度、长期储存稳定性和大规模生产的重现性。(C)2005 Elsevier B.V.保留所有权利。
A stable, freeze-dried formulation consisting of a core-shell-type polyplex with a poly(ethylene glycol) (PEG) shell (polyplex micelles) was prepared from a polyion complex of plasmid DNA (pDNA) and thiolated PEG-poly(L-lysine) block copolymers. The use of lyoprotectants was avoided by crosslinking the core with disulfide bonds. The crosslinked polyplex micelles (CPMs) showed excellent stability during freeze-drying and reconstitution processes, which is in sharp contrast with the formation of visible agglomerates from the non-crosslinked polyplex micelles (NCPMs) after a similar process. A thiolation degree higher than 13 % of the lysine residues was required to achieve sufficient tolerability of the CPMs during the freeze-drying/reconstitution cycle. Dynamic light scattering measurements and atomic force microscopy observations demonstrated that the original size and shape of the CPMs with a thiolation degree of higher than 13% were maintained even after the freeze-drying. Furthermore, the CPMs reconstituted from the freeze-dried state achieved a transfection efficiency as high as that of the original samples. The intravenous injection of the CPM with a thiolation degree of 37% into mice via the orbital vein led to an appreciably uniform gene expression of a yellow fluorescence protein variant (Venus) in the liver, while no gene expression was observed in the case of the free pDNA injection. The procedure of disulfide crosslinking of the polyplex micell core allows the preparation of non-viral gene vectors as a powder formulation without the use of any lyoprotectants. This achievement is certainly useful for pharmaceutical applications and exhibits many advantages, including easy concentration adjustments of dosing samples, long-term storage stability, and large-scale production reproducibility. (c) 2005 Elsevier B.V. All rights reserved.