Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore.
Biomaterials. 2012 Sep;33(26):6284-91. doi: 10.1016/j.biomaterials.2012.05.033. Epub 2012 Jun 12.
In this study, a number of KALA-based α-helical peptides were designed and synthesized as non-viral gene carriers. The effects of lysine and histidine residues in the pendant arms and cell-binding RGD motif on DNA binding, particle size, zeta potential, cytotoxicity and gene expression efficiency were first explored. Increasing the lysine and histidine residues reduced particle size and increased zeta potential of DNA complexes, leading to greater gene expression efficiency. In addition, the introduction of RGD group further improved gene expression level. The peptide with optimal compositions, RGDN(3)K(6)H(3)CKHLAKALAKALAC (RC29), was then oligomerized to form di-, tri- and tetra-RC29 via disulfide linkage. Upon oligomerization, RC29 attained a 3-dimensional long α-helical structure with pendant arm(s) extending transversally outwards. Each arm contains a cell-adhesion motif (RGD), DNA-binding and endosome-buffering domains(.) The α-helicity of the oligomerized peptides was evaluated by circular dichroism (CD) spectroscopy, which showed that an increased oligomerization degree led to a stronger α-helical structure. These peptides form complexes with DNA efficiently. The minimum size and maximum zeta potential of tri-RC29/DNA complexes was about 200 nm and 32.5 mV, respectively. In comparison, RC29 formed DNA complexes with a similar zeta potential, but particle size was significantly larger (355 nm). DNA complexes formed at pH 7.0 yielded higher gene expressions than those formed at pH 5.5 and 6.5. Among all the oligomerized peptides, tri-RC29 provided the highest gene expression efficiency, and its peak luciferase level was 1.5 times higher than that yielded by PEI at its optimal N/P ratio (i.e. 10). Moreover, oligomerized RC29/DNA complexes were less cytotoxic than PEI/DNA complexes. These α-helical peptides can be promising carrier for delivery of therapeutic genes in the treatment of genetic disorders.
在这项研究中，设计并合成了一系列基于 KALA 的 α-螺旋肽作为非病毒基因载体。首先研究了侧臂中的赖氨酸和组氨酸残基以及细胞结合 RGD 基序对 DNA 结合、颗粒大小、zeta 电位、细胞毒性和基因表达效率的影响。增加赖氨酸和组氨酸残基会减小 DNA 复合物的颗粒大小并增加 zeta 电位，从而提高基因表达效率。此外，引入 RGD 基团进一步提高了基因表达水平。然后，将具有最佳组成的肽 RGDN(3)K(6)H(3)CKHLAKALAKALAC (RC29) 寡聚形成二聚体、三聚体和四聚体通过二硫键连接。寡聚化后，RC29 形成了具有横向向外延伸的侧臂的 3 维长 α-螺旋结构。每个臂都包含一个细胞黏附基序（RGD）、DNA 结合和内涵体缓冲域。圆二色性（CD）光谱学评估了寡聚肽的 α-螺旋性，结果表明增加的寡聚化程度导致更强的 α-螺旋结构。这些肽可以有效地与 DNA 形成复合物。三 RC29/DNA 复合物的最小尺寸和最大 zeta 电位分别约为 200nm 和 32.5mV。相比之下，RC29 形成的 DNA 复合物具有相似的 zeta 电位，但粒径明显更大（355nm）。在 pH7.0 下形成的 DNA 复合物比在 pH5.5 和 6.5 下形成的复合物具有更高的基因表达。在所有寡聚肽中，三 RC29 提供了最高的基因表达效率，其峰值荧光素酶水平比其最佳 N/P 比（即 10）下的 PEI 高 1.5 倍。此外，寡聚 RC29/DNA 复合物的细胞毒性比 PEI/DNA 复合物低。这些 α-螺旋肽可作为治疗基因传递的有前途的载体，用于治疗遗传疾病。
