Department of Biochemistry, Case Western Reserve University Schoolof Medicine, Cleveland, Ohio 44106, USA.
J Drug Target. 2012 Sep;20(8):678-90. doi: 10.3109/1061186X.2012.712128.
DNA nanoparticles (DNA NPs), which self-assemble from DNA plasmids and poly-L-lysine (pLL)-polyethylene glycol (PEG) block copolymers, transfect several cell types in vitro and in vivo with minimal toxicity and immune response. To further enhance the gene transfer efficiency of DNA NP and control its tropism, we established a strategy to efficiently attach peptide ligands to DNA NPs. The non-covalent biotin-streptavidin (SA) interaction was used for ligand conjugation to overcome problems associated with covalent conjugation methods. A fusion protein of SA with the HIV-1 TAT peptide was cloned, expressed, purified and attached to biotinylated DNA NPs. A modified SA system with tetrameric structure but monovalent biotin binding capacity was adopted and shown to reduce the aggregation of biotinylated DNA NPs compared to neutravidin. Compared to unmodified DNA NPs, TAT modified DNA NPs significantly enhanced in vitro gene transfer, particularly at low DNA concentrations. Studies of cellular uptake and cellular distribution of the DNA NPs indicated that attaching TAT enhanced binding of DNA NPs to cell surface but not internalization at high DNA concentrations. In vivo studies showed that TAT modified DNA NPs mediated equal level of gene transfer to the mouse airways via the luminal route compared to unmodified DNA NPs.
DNA 纳米颗粒（DNA NPs）可自组装形成 DNA 质粒和聚 L-赖氨酸（pLL）-聚乙二醇（PEG）嵌段共聚物，具有低毒性和免疫原性，可在体外和体内转染多种细胞类型。为了进一步提高 DNA NP 的基因转移效率并控制其趋向性，我们建立了一种有效将肽配体连接到 DNA NP 的策略。非共价生物素-链霉亲和素（SA）相互作用用于配体连接，以克服与共价连接方法相关的问题。克隆、表达、纯化了与 HIV-1 TAT 肽融合的 SA 融合蛋白，并将其连接到生物素化的 DNA NP 上。采用了具有四聚体结构但单价生物素结合能力的改良 SA 系统，与链霉亲和素相比，该系统可减少生物素化 DNA NP 的聚集。与未修饰的 DNA NP 相比，TAT 修饰的 DNA NP 显著增强了体外基因转移，特别是在低 DNA 浓度下。对 DNA NP 的细胞摄取和细胞分布的研究表明，连接 TAT 增强了 DNA NP 与细胞表面的结合，但在高 DNA 浓度下并未促进内化。体内研究表明，TAT 修饰的 DNA NP 通过管腔途径介导与未修饰的 DNA NP 相同水平的基因转移到小鼠气道。
