目的:这项研究的目的是开发一种基于脂肽的通用基因载体,该载体能够以最小的细胞毒性将遗传物质递送到靶细胞中。
方法:两个脂肽分子,棕榈酰-CKKHH和棕榈酰-CKKHH-YGRKKRRQRRR-PKKKRKV,使用固相肽合成合成并作为转染剂进行评估。脂肽的物理化学表征包括DNA移位迁移率测定,粒度测量,和透射电子显微镜(TEM)分析。使用MTT测定在CHO-K1和HepG2细胞中评估细胞毒性,通过评估绿色荧光蛋白编码基因的表达来确定转染效率。
结果:我们的研究结果表明,脂肽可以结合,冷凝,并保护DNA免受DNA酶降解。包含YGRKKRRQRRR序列,转录反式激活剂,和PKKKRKV序列,一个核定位信号,赋予理想的属性。基于脂肽的TAT-NLS/DNA纳米颗粒在6-8°C储存时表现出长达20天的稳定性,显示具有约120nm的紧凑尺寸的均匀性。此外,与聚-L-赖氨酸相比,脂肽表现出更低的细胞毒性。转染实验表明,由脂肽介导的蛋白质表达发生在4.0至8.0的电荷比下。
结论:这些结果表明,脂肽,由棕榈酰基烷基链和TAT和NLS序列组成,可以有效地浓缩和保护DNA,形成稳定均匀的纳米粒子,并表现出作为具有最小细胞毒性的潜在基因载体的有希望的特征。
OBJECTIVE: The objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity.
METHODS: Two
lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene.
RESULTS: Our findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties.
Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6-8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the
lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0.
CONCLUSIONS: These results indicate that the
lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.