Beijing Institute of Transfusion Medicine, Beijing, People's Republic of China.
J Gene Med. 2012 Apr;14(4):241-50. doi: 10.1002/jgm.2609.
Melittin is a commonly used cell-penetrating peptide (CPP) for improving branched polyethylenimine (BPEI)-mediated gene transfection. However, its application is limited owing to the cytotoxicity generated by the lytic activity at neutral pH. In the present study, we report two truncated peptides from melittin and florae with improved transfection efficiency.
Two truncated peptides consisting of 1-20 residues of melittin (MT20) and florae (FL20) were synthesized. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. The membrane-lytic activity of the peptides and their potency in enhancing cellular uptake of calcein were evaluated. The peptides and BPEI mixtures were mixed with plasmid DNA to prepare peptide/BPEI/DNA complexes. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined.
CD analysis and structure observation showed that the truncated peptides have α-helical conformation, which was necessary for penetrating activity. The truncated peptides exhibited several advantages than their parent peptides: (i) they showed higher hemolytic potency in acidic pH but lower lytic activity than their parent peptides in neutral pH; (ii) enhanced calcein efficiently release from both early and late endosome; (iii) they did not affect the DNA-binding affinity of BPEI and the physicochemical characteristics of BPEI/DNA complexes. Moreover, the peptides could increase BPEI-mediated transfection efficiency in different cell lines (293FT, B16F10 and CHO-K1) by simply mixing with BPEI, without causing cytotoxicity.
The results obtained in the present study indicate that the truncated peptides with higher endosomal disrupting activity were better enhancers for increasing transfection efficiency.
蜂毒素是一种常用的细胞穿透肽（CPP），可提高支化聚乙烯亚胺（BPEI）介导的基因转染。然而，由于在中性 pH 值时的溶细胞活性，其应用受到限制。在本研究中，我们报道了两种从蜂毒素和植物中截短的肽，它们具有提高转染效率的作用。
合成了由蜂毒素（MT20）和植物 1-20 个残基组成的两种截短肽。圆二色性（CD）光谱用于分析肽的二级结构。评估了肽的膜裂解活性及其增强 calcein 细胞摄取的能力。将肽与 BPEI 混合物与质粒 DNA 混合制备肽/BPEI/DNA 复合物。测量复合物的物理化学特性，并确定肽对 BPEI 介导的转染的影响。
CD 分析和结构观察表明，截短的肽具有α-螺旋构象，这对于穿透活性是必需的。与亲本肽相比，截短的肽具有以下几个优点：（i）它们在酸性 pH 值下表现出更高的溶血效力，但在中性 pH 值下比亲本肽的裂解活性低；（ii）增强了 calcein 从早期和晚期内涵体中的有效释放；（iii）它们不影响 BPEI 的 DNA 结合亲和力和 BPEI/DNA 复合物的物理化学特性。此外，这些肽通过与 BPEI 简单混合，即可增加不同细胞系（293FT、B16F10 和 CHO-K1）中的 BPEI 介导的转染效率，而不会引起细胞毒性。
本研究结果表明，具有更高内涵体破坏活性的截短肽是提高转染效率的更好增强剂。
