肝素修饰金纳米粒子的制备及表征

Preparation and characterization of heparin-modified gold nanoparticles

背景:大分子修饰的金纳米粒子可被应用于治疗学或诊断学等其他方面,但是大多数报道多限于制备蛋白质、DNA及小分子糖修饰的金纳米粒子方面,对于制备多糖修饰的金纳米粒子的研究尚不深入。目的:制备一种肝素修饰的金纳米粒子,考察工艺条件并对其粒径及紫外吸收光谱进行观察。设计、时间及地点:对比观察实验,于2007-04/2008-09在武汉理工大学化工学院生物技术制药实验室完成。材料:制备金纳米粒子并在4℃保存供实验用。方法:将肝素钠用亚硝酸降解法降解,得到还原末端为醛基的肝素,分别溶解于二甲亚砜、含0.05mL,0.5mL冰醋酸的二甲亚砜溶液,再与对巯基苯胺发生还原胺化反应,用氰基硼氢化钠作还原剂,得到带巯基的肝素衍生物。另取样品溶解于4支含0.5mL冰醋酸的二甲亚砜溶液的瓶内,再加入对巯基苯胺和氰基硼氢化钠,分别反应2,4,6,24h。将得到的肝素衍生物加入到制备好的金纳米粒子溶液中,肝素以Au-S化学键合的方式定向固定在金纳米粒子的表面。主要观察指标:①肝素修饰前后金纳米粒子的紫外光谱最大吸收波长。②肝素修饰前后金纳米粒子的粒径大小。结果:制备金纳米粒子时,当柠檬酸钠和氯金酸物质的量比为6∶1时,纳米粒子粒径均一。通过还原胺化反应制备端基为巯基的肝素时,加入冰醋酸的量越多,反应产率越高。反应6h内,反应时间越长,其反应产率越高,24h的反应产率虽比6h的略有提高,但差别不大,说明6h反应已经基本完全。将肝素以Au-S化学键合的方式定向固定在金纳米粒子的表面,金纳米粒子的粒径为10nm,紫外最大吸收为522nm;肝素修饰的金纳米粒子的粒径为20nm,紫外最大吸收为529nm。结论:制备了粒径均一的肝素修饰的金纳米粒子,并且粒径大小对纳米粒子的紫外最大吸收有影响。还原胺化反应的最佳反应条件是采用浓度比为8∶1的二甲亚砜/冰醋酸混合溶液作为溶剂,反应时间为6h。

BACKGROUND： Macromolecules modified gold nanoparticles can be used in therapeutics, diagnostics, and other aspects, but most reports mainly focuses on the preparation of protein, DNA and small molecules of sugar modified gold nanoparticles, and the research on polysaccharide modified gold nanoparticles is superficial. OBJECTIVE： To prepare heparin-modified gold nanoparticles, studying their particle size and ultraviolet absorption spectra, and to determine the optimum reaction condition. DESIGN, TIME AND SETTING： The comparative observation of the experiment was performed at the Biotechnology Pharmaceutical Laboratory of School of Chemical Engineering in Wuhan University of Technology from April 2007 to September 2008. MATERIALS： Gold nanoparticles were prepared and preserved at 4℃. METHODS： Heparin with an aldehyde group （HEP-CHO） as reducing end was prepared by nitrous acid degradation method. HEP-CHO was further reacted with dimethyl sulfoxide solution containing 0.05 mL and 0.5 mL glacial acetic acid to synthesize heparin with thiol-group （HEP-SH） through reductive amination reaction. Heparin was then orientated immobilized in four bottles of dimethyl sulfoxide solution containing 0.5 mL glacial acetic acid by adding the thiol-group of HEP-SH and sodium cyanoborohydride for 2, 4, 6, and 24 hours. The obtained products were added in gold nanoparticle solution, and the heparin was fixed on the surface of gold nanoparticles by Au-S bonds. MAIN OUTCOME MEASURES： （~ Maximum ultraviolet absorbance of gold nanoparticles before and after heparin modified; （D Average grain size of gold nanoparticles and heparin-modified gold nanoparticles. RESULTS： Gold nanoparticles with good particle size were prepared by using a rate of sodium citrate to chloroauric acid was 6：1 （mol/mol）. The reductive amination reaction yield was higher when the more acetic acid was added to the solvent. The products were increased within 6 hours and 24 hours, but the difference was not significant. The average grain size and maximum ultraviolet absorbance of gold nanoparticles were 10 nm and 522 nm, and those of heparin-modified gold nanoparticles were 20 nm and 529 nm. CONCLUSION： Heparin-modified gold nanoparticles with good particle size are prepared, and the size can effect on the maximum ultraviolet absorbance. The optimum reaction condition for heparin immobilization is using a rate of dimethyl sulfoxide to acetic acid is 8：1 （mol/mol） as solvent of heparin, and the reaction time is 6 hours.