貴州醫(yī)科大學藥學院，貴州省特色天然藥物資源高效利用工程中心，貴州省高等學校天然藥物藥理與成藥性評價特色重點實驗室，貴州醫(yī)科大學-貴陽市聯(lián)合重點實驗室，天然藥物資源優(yōu)效利用重點實驗室，貴州貴陽561113摘要：目的 研究膽固醇（CH）對脂微球（LM）與人血清白蛋白（HSA）形成蛋白暈的影響。方法 采用高速剪切-高壓均質(zhì)兩步乳化法制備LM及含CH的CH@LM后，分別將相同體積但不同濃度（8、12、24、36 μmol·L^-1）的HSA溶液與等體積不同質(zhì)量濃度（10.76、15.37、26.90、35.87、53.80 mg·mL^-1）的LM及CH@LM（以LM計）用恒溫震蕩器孵育不同時間，采用尺寸排阻色譜法分離出含有HSA蛋白暈的LM-HSA和CH@LM-HSA復合物以及過剩的HSA。通過粒徑儀檢測粒徑及聚合物分散性指數(shù)（PDI），紫外可見分光光度計檢測蛋白暈復合物的紫外圖譜，熒光酶標儀檢測蛋白暈復合物熒光圖譜，分子對接研究油相中輔料的主要成分與HSA的結(jié)合能、油相中各輔料之間的結(jié)合能，并采用考馬斯亮藍染色對蛋白暈復合物進行表征。結(jié)果 LM、CH@LM自身穩(wěn)定性較好，LM與HSA之間存在相互作用，所形成復合物的粒徑及PDI具有時間及濃度相關(guān)性；加入CH后改變了復合物的粒徑隨時間的變化模式，同時減弱了由于LM濃度變化引起的與蛋白質(zhì)之間相互作用的變化。加入CH后HSA自身熒光淬滅程度增強，最大吸收波長藍移程度增加，表明加入CH后增強了LM與HSA的相互作用程度。CH與HSA的分子對接結(jié)合能絕對值最高，且有CH的油相中各輔料分子與分子間對接結(jié)合能均高于無CH參與組；LM-HSA及CH@LM-HSA均保留了HSA的特征條帶。結(jié)論 CH增強了LM對HSA的吸附能力，CH與其他輔料分子和蛋白具有更強的結(jié)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

國家自然科學基金資助項目（82260827）;貴州省科技計劃項目黔科合基礎(chǔ)-ZK［2022］380;貴州省科技創(chuàng)新基地，黔科合中引地［2023］003;貴州省高層次創(chuàng)新型人才十層次人才黔科合平臺人才-GCC［2023］048;貴州醫(yī)科大學國家自然科學基金培育項目（20NSP050）;貴州醫(yī)科大學高層次人才科研啟動基金項目校博合J字（2023）026號