贵州医科大学药学院，贵州省特色天然药物资源高效利用工程中心，贵州省高等学校天然药物药理与成药性评价特色重点实验室，贵州医科大学-贵阳市联合重点实验室，天然药物资源优效利用重点实验室，贵州贵阳561113摘要：目的 研究胆固醇（CH）对脂微球（LM）与人血清白蛋白（HSA）形成蛋白晕的影响。方法 采用高速剪切-高压均质两步乳化法制备LM及含CH的CH@LM后，分别将相同体积但不同浓度（8、12、24、36 μmol·L^-1）的HSA溶液与等体积不同质量浓度（10.76、15.37、26.90、35.87、53.80 mg·mL^-1）的LM及CH@LM（以LM计）用恒温震荡器孵育不同时间，采用尺寸排阻色谱法分离出含有HSA蛋白晕的LM-HSA和CH@LM-HSA复合物以及过剩的HSA。通过粒径仪检测粒径及聚合物分散性指数（PDI），紫外可见分光光度计检测蛋白晕复合物的紫外图谱，荧光酶标仪检测蛋白晕复合物荧光图谱，分子对接研究油相中辅料的主要成分与HSA的结合能、油相中各辅料之间的结合能，并采用考马斯亮蓝染色对蛋白晕复合物进行表征。结果 LM、CH@LM自身稳定性较好，LM与HSA之间存在相互作用，所形成复合物的粒径及PDI具有时间及浓度相关性；加入CH后改变了复合物的粒径随时间的变化模式，同时减弱了由于LM浓度变化引起的与蛋白质之间相互作用的变化。加入CH后HSA自身荧光淬灭程度增强，最大吸收波长蓝移程度增加，表明加入CH后增强了LM与HSA的相互作用程度。CH与HSA的分子对接结合能绝对值最高，且有CH的油相中各辅料分子与分子间对接结合能均高于无CH参与组；LM-HSA及CH@LM-HSA均保留了HSA的特征条带。结论 CH增强了LM对HSA的吸附能力，CH与其他辅料分子和蛋白具有更强的结合能力可能是主要原因。

Objective To study effect of cholesterol (CH) on the formation of protein coronas between lipid microspheres (LM) and human serum albumin (HSA). Methods After preparing LM and CH@LM using a two-step emulsification method involving highspeed shear-high-pressure homogenization, HSA solution with the same volume but different concentrations (8, 12, 24, 36 μmol·L^-1) and LM and CH@LM(calculated in LM) with the same volume and different mass concentrations (10.76, 15.37, 26.90, 35.87, 53.80 mg·mL^-1) were incubated with constant temperature oscillators for different times, respectively. The LM-HSA and CH@LMHSA complexes containing HSA protein coronas and excess HSA were isolated by size-exclusion chromatography. Particle size and polymer dispersion index (PDI) were detected by particle size meter, ultraviolet visible spectrophotometry was used to detect the ultraviolet spectrum of the protein coronas complex, fluorescence enzyme spectrometer was used to detect the fluorescence spectrum of the protein coronas complex, and molecular interconnection was conducted to study the binding energy between the main components of excipients in the oil phase and HSA, as well as the binding energy between excipients in the oil phase. The protein coronas complex was characterized by Coomassie bright blue staining. Results Both LM and CH@LM demonstrate excellent inherent stability. An interaction between LM and HSA is observed, leading to the formation of complexes with particle size and PDI that are dependent on both time and concentration. The introduction of CH further enhances the fluorescence quenching effect on HSA itself, while also increasing the degree of blue shift in maximum absorption wavelength, indicating an intensified interaction between LM and HSA upon CH addition. The binding energy between CH and HSA exhibits the highest absolute value, surpassing that without CH involvement, thereby resulting in higher intermolecular binding energy when CH is present. Both LM-HSA and CH@LM-HSA retain characteristic bands associated with HSA. Conclusion CH enhanced the adsorption capacity of LM to HSA, and the stronger binding ability of CH with other excipient molecules and proteins may be the main reason.

R943

国家自然科学基金资助项目（82260827）;贵州省科技计划项目黔科合基础-ZK［2022］380;贵州省科技创新基地，黔科合中引地［2023］003;贵州省高层次创新型人才十层次人才黔科合平台人才-GCC［2023］048;贵州医科大学国家自然科学基金培育项目（20NSP050）;贵州医科大学高层次人才科研启动基金项目校博合J字（2023）026号