The Center for Nanomedicine, The Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21231, USA.
Biomaterials. 2012 Mar;33(7):2361-71. doi: 10.1016/j.biomaterials.2011.11.080. Epub 2011 Dec 17.
Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-L-lysine (PEG-CK(30)), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-L-histidine was inserted between PEG and poly-L-lysine to form a triblock copolymer system, PEG-CH(12)K(18). Inclusion of poly-L-histidine increased the buffering capacity of PEG-CH(12)K(18) to levels comparable with branched polyethyleneimine. PEG-CH(12)K(18) compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK(30) DNA nanoparticles. PEG-CH(12)K(18) DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH(12)K(18) DNA nanoparticles improved the in vitro gene transfer by ~20-fold over PEG-CK(30) DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ~3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.
高度紧凑的 DNA 纳米颗粒由单分子质粒 DNA 与聚乙二醇和聚-L-赖氨酸的嵌段共聚物（PEG-CK(30)）压缩而成，在鼻腔内的人类基因治疗临床试验中显示出了相当大的前景，但可能不太能够转染缺乏表面核仁素的细胞。为了解决这一潜在的缺点，我们设计了 pH 响应性 DNA 纳米颗粒，通过核仁素非依赖性途径介导基因转移。聚-L-组氨酸被插入聚乙二醇和聚-L-赖氨酸之间，形成三嵌段共聚物系统，PEG-CH(12)K(18)。聚-L-组氨酸的插入增加了 PEG-CH(12)K(18)的缓冲能力，使其与支化聚乙烯亚胺相当。PEG-CH(12)K(18)将 DNA 压缩成棒状 DNA 纳米颗粒，其形态和胶体稳定性与 PEG-CK(30) DNA 纳米颗粒相似。PEG-CH(12)K(18) DNA 纳米颗粒通过网格蛋白依赖性内吞作用进入缺乏表面核仁素的人支气管上皮细胞 (BEAS-2B)，随后进行内体溶酶体处理。尽管通过降解性内体溶酶体途径运输，但 PEG-CH(12)K(18) DNA 纳米颗粒将体外基因转移提高了约 20 倍，体内基因转移到 BALB/c 小鼠的肺气道提高了约 3 倍，同时保持了良好的毒性特征。这些结果代表了朝着基于高度紧凑的 DNA 纳米颗粒的高效肺部基因传递平台的合理发展迈出了重要的一步。
