貴州醫(yī)科大學(xué)藥學(xué)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(xué)校天然藥物藥理與成藥性評(píng)價(jià)特色重點(diǎn)實(shí)驗(yàn)室，貴州醫(yī)科大學(xué)-貴陽(yáng)市聯(lián)合重點(diǎn)實(shí)驗(yàn)室，天然藥物資源優(yōu)效利用重點(diǎn)實(shí)驗(yàn)室，貴州貴陽(yáng)561113摘要：目的 研究膽固醇（CH）對(duì)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(jì)）用恒溫震蕩器孵育不同時(shí)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(fù)合物以及過(guò)剩的HSA。通過(guò)粒徑儀檢測(cè)粒徑及聚合物分散性指數(shù)（PDI），紫外可見分光光度計(jì)檢測(cè)蛋白暈復(fù)合物的紫外圖譜，熒光酶標(biāo)儀檢測(cè)蛋白暈復(fù)合物熒光圖譜，分子對(duì)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(lán)染色對(duì)蛋白暈復(fù)合物進(jìn)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(fù)合物的粒徑及PDI具有時(shí)間及濃度相關(guān)性；加入CH后改變了復(fù)合物的粒徑隨時(shí)間的變化模式，同時(shí)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(qiáng)，最大吸收波長(zhǎng)藍(lán)移程度增加，表明加入CH后增強(qiáng)了LM與HSA的相互作用程度。CH與HSA的分子對(duì)接結(jié)合能絕對(duì)值最高，且有CH的油相中各輔料分子與分子間對(duì)接結(jié)合能均高于無(wú)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(qiáng)了LM對(duì)HSA的吸附能力，CH與其他輔料分子和蛋白具有更強(qiáng)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(guó)家自然科學(xué)基金資助項(xiàng)目（82260827）;貴州省科技計(jì)劃項(xiàng)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(tái)人才-GCC［2023］048;貴州醫(yī)科大學(xué)國(guó)家自然科學(xué)基金培育項(xiàng)目（20NSP050）;貴州醫(yī)科大學(xué)高層次人才科研啟動(dòng)基金項(xiàng)目校博合J字（2023）026號(hào)