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目的:将小分子靶向肽RGD(Arg-Gly-Asp)偶联到壳聚糖(CS)上,并包载质粒DNA(pDNA),制成一种具有靶向性的壳聚糖载基因纳米粒。方法:将RGD肽上的羧基和CS上的氨基通过酰化反应发生偶联,运用红外(FT-IR)和元素分析对RGD偶联壳聚糖(CS-RGD)的化学结构进行确证;采用复凝聚法制备CS-RGD/pDNA纳米粒(CS-RGD/pDNA);应用凝胶阻滞实验和DNA酶(DNase I)降解实验考察CS-RGD对pDNA的复合和保护能力;通过激光粒度仪和原子力显微镜对纳米粒的粒径分布和形态进行考察。结果:CS和RGD肽通过酰胺键偶联;CS-RGD/pDNA在N/P≥2时完全复合,在N/P≥4时具有抗DNase I酶降解能力,N/P=2～30的CS-RGD/pDNA复合物粒径在90～260 nm之间,Zeta电位在4～39 mV之间,原子力显微镜结果证明复合物为类球形且分布良好。具有良好的稳定性和易于进入细胞的性质。结论:CS-RGD是一种制备工艺简单,具有应用前景的非病毒基因载体。
OBJECTIVE: To couple the small molecule targeting peptide RGD (Arg-Gly-Asp) to chitosan (CS) and encapsulate plasmid DNA (pDNA) into a targeted chitosan- Nanoparticles. Methods: The carboxyl groups on RGD peptide and the amino groups on CS were coupled by acylation. The chemical structure of RGD-coupled chitosan (CS-RGD) was confirmed by FT-IR and elemental analysis. The CS-RGD / pDNA nanoparticles (CS-RGD / pDNA) were prepared by the complex coacervation method. The effects of CS-RGD on the pDNA complexation and protection were investigated by gel retardation assay and DNase I degradation assay. And atomic force microscopy of nanoparticles size distribution and morphology were investigated. Results: CS and RGD peptide were coupled by amide bond. CS-RGD / pDNA completely complexed when N / P≥2 and anti-DNase I enzyme degradation ability at N / P≥4, N / P = 2-30 The particle size of CS-RGD / pDNA complex ranged from 90 to 260 nm and the Zeta potential ranged from 4 to 39 mV. The results of atomic force microscopy showed that the complex was spheroidal and well-distributed. Has good stability and easy to enter the cell nature. Conclusion: CS-RGD is a non-viral gene vector with simple preparation process and promising application.