白藜芦醇白蛋白纳米粒的制备及其抗卵巢癌细胞增殖作用的研究

以牛血清白蛋白(BSA)为载体, 用去溶剂化-化学交联法制备白藜芦醇白蛋白纳米粒(RES-BSANP). 以原子力学显微镜(AFM)观察其形态, 用高效液相色谱法(HPLC)对制备的纳米微粒进行分析. 采用四甲基偶氮唑盐微量酶反应比色法(MTT)及流式细胞技术(FCM)比较RES-BSANP和RES对卵巢癌SKOV3细胞的抗增殖活性及对细胞周期和凋亡的影响. 结果表明, 获得的RES-BSANP纳米粒的平均粒径为400～500 nm, 表面光滑, 12 mg纳米粒中RES载药量为4.077 mg, 包封率33.97％, 24 h内的稳定性好, 水溶性较RES显著提高. 二者的抗肿瘤增殖作用呈剂量依赖性, 中高浓度组纳米粒组的抗增殖活性及凋亡细胞比率显著提高. 两种药物均使细胞周期阻滞于G0/G1+S期, 纳米组使进入S期细胞比率明显增加, 表明白藜芦醇白蛋白纳米粒在抗卵巢癌细胞增殖方面有广阔的应用前景.     

半胱氨酸对高效液相色谱法测定多西紫杉醇白蛋白纳米粒含量的影响

建立多西紫杉醇(docetaxel,DOC)人血清白蛋白纳米粒(human serum albumin nanoparticles,HSA-NP)中药物含量测定的方法.利用半胱氨酸(cysteine,Cys)还原人血清白蛋白中交联的二硫键,释放HSA-NP中包裹的药物,再利用HPLC测定药物含量.结果表明,在本实验条件下Cys及制剂中辅料对DOC的含量测定无干扰.1 μg/L Cys、37 ℃孵化30 min,最后用乙腈沉淀蛋白的方法可以完全测定出制剂中DOC含量.     

脂质组成对硫辛酸脂质纳米粒的制备及其稳定性的影响

采用高压均质法制备硫辛酸(ALA)脂质纳米粒(ALA-NLC),确定了负载ALA的最佳体系。通过高效液相色谱、动态光散射、流变等测试研究了脂质成分、含量对ALA负载、ALA-NLC稳定性和分散液流变性质的影响,并用原子力显微镜观察了ALA-NLC的形貌。结果表明,脂质/ALA质量比在4.5∶1～6∶1之间,得到的脂质纳米粒分散均匀,颗粒规则;脂质中液态油脂的增加有利于保持ALA-NLC的粒径,储存稳定性和分散液的低粘度,并得到两个有望用于食品工业的ALA-NLC配方。     

光谱-分子模拟法分析杨梅素与人血清白蛋白的分子作用机制

在模拟生理条件下,用多种光谱法结合分子对接法测定了杨梅素(MY)与人血清白蛋白(HSA)的相互作用.研究结果表明,MY能够明显猝灭HSA的荧光,MY与HSA的相互作用为复合式静态结合过程,结合强度较强.热力学和分子对接结果表明,MY与HSA是自发结合的,维持MY与HSA的相互作用力主要是氢键和范德华力.能量转移结果表明...     

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C₄₄), 83% carbamylated (C₈₃) and 85% acetylated (A₈₅) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C₈₃ and A₈₅ HSAs while C₄₄ HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (K_SV) and bimolecular quenching rate constant (k_q) suggested, NA-HSA complex formation, binding constant (K_a) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the K_a value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.     

Study on the interactions of sulfonylurea antidiabetic drugs with normal and glycated human serum albumin by capillary electrophoresis‐frontal analysis

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis‐frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis‐frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.     

Identification of modification sites on human serum albumin and human hemoglobin adducts with houttuynin using liquid chromatography coupled with mass spectrometry

Houttuynin, a β‐keto aldehyde compound, is the major active ingredient in herba houttuyniae injection. The injection was once used as an anti‐inflammatory drug associated with occasional serious hypersensitivity reactions in the clinic, which were proposed to be related to the formation of protein adducts. N^α‐Boc‐lysine, FEEM and IVTNTT were used as model amino acids or peptides containing one nucleophilic residue to investigate adduct types by liquid chromatography coupled with ion trap mass spectrometry (LC/MSⁿ) and high‐resolution quadrupole time‐of‐flight mass spectrometry (Q‐TOF MS). These adducts were respectively characterized as Schiff bases formed by 1:1 reaction of houttuynin with lysine or N‐terminal residue and pyridinium adducts by 2:1 reaction. LC/MSⁿ analysis of trypsin digests of HSA/Hb incubations with houttuynin revealed that houttuynin‐modified HSA adducts were formed mainly at N‐terminal amino acid and lysine residues, specifically at Lys‐212, Lys‐414 and Lys‐525 for Schiff base adducts, and at Lys‐414 and Lys‐432 for pyridinium adducts, and houttuynin adducted more readily with N‐terminal valine of the α‐ and β‐chains in Hb and lysine amine (Lys‐62) of the β‐chain for Schiff base adducts. The results showed the direct modification of houttuynin to HSA/Hb in vitro, which was speculated to be responsible for the adverse reactions induced by houttuyniae injection. Copyright © 2012 John Wiley & Sons, Ltd.     

Cu(II), Fe(III)与人血清白蛋白相互作用的荧光光谱研究

通过研究Cu(II),Fe(III)对人血清白蛋白(HSA)内源荧光的猝灭,探讨了Cu(II),Fe(III)与人血清白蛋白的结合机理。基于Forster非辐射能量转移机理。获得了人血清白蛋白第一类Cu(II)结合部位与214位色氨酸残基间的距离为1.8nm,并讨论了Cu(II),Fe(III)与HSA结合的差异。     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

计算模拟与光谱法研究4-羟基-2, 2＇, 3, 4＇-四溴二苯醚与人血清白蛋白的相互作用

利用分子模拟、荧光光谱、紫外吸收光谱等方法,研究了4-羟基-2,2’,3,4’-四溴二苯醚(4-OHBDE-42)与人血清白蛋白(HSA)的相互作用。三维荧光分析表明,4-OH-BDE-42的存在降低了HSA的荧光强度,且使HSA的微环境和构象发生变化。荧光光谱和紫外吸收光谱显示,4-OH-BDE-42与HSA结合后显著猝灭了HSA的内源性荧光,猝灭机制为静态猝灭与非辐射能量转移。结合常数Ka106L·mol-1,表明两者的结合作用较强,结合距离r为3.66nm。根据热力学参数分析,ΔH0,ΔS0,即4-OH-BDE-42与HSA之间结合的主要作用力为疏水作用,这与分子对接、结合自由能分析结论一致。结合自由能贡献分析表明,LYS199、GLU292、ARG257、ARG218、ALA291、HIS242为4-OH-BDE-42与HSA结合的关键氨基酸残基。     

Au@4-硝基苯硫酚@Ag@牛血清白蛋白内标化表面增强拉曼散射探针的制备及其在细胞拉曼成像中的应用

制备了Au@4-硝基苯硫酚@Ag内标化表面增强拉曼散射（SERS）探针,进一步以牛血清白蛋白（BSA）置换探针表面的稳定剂十六烷基三甲基溴化铵（CTAB）,发展了Au@NT@Ag@BSA内标化SERS探针。Au@NT@Ag@BSA探针保留了原探针的单分散性和高灵敏度,同时显著提高了信号稳定性和生物相容性。进一步将Au@NT@Ag@BSA探针和SMMC7721肺癌细胞共孵育,实现了细胞的探针标记和拉曼光谱成像。Au@NT@Ag@BSA内标化SERS探针在活体生物成像等方面展示了良好的应用潜力。     

Electrochemical behavior of Ru(H_2bpp)_2(PF_6)_2 and its interaction with bovine serum albumin(BSA)

In this paper,it was found that Ru（H₂bpp）₂（PF₆）2（H₂bpp = 2,6-bis（pyrazol-3-yl）pyridine） complex had excellent electrochemical activity at the carbon paste electrode in the buffer solution of Tris-HCl（pH 7.0） with a couple reversible redox peaks at 0.296 V and 0.348 V,respectively.Voltammetry was used to investigate the electrochemical behavior of Ru（H₂bpp）₂（PF₆）₂ and the interaction between Ru（H₂bpp）₂（PF₆）₂ and bovine serum albumin（BSA）.In the present of BSA,the oxidation peak current of Ru（H₂bpp）₂（PF₆）₂ complex was decreased linearly and the decrease of oxidation peak current of Ru（H₂bpp）₂（PF₆）₂ is proportional to BSA concentration from 0.1 to 2.5 mg/L with a detection limit 0.02 mg/L.     

Quantitation of urapidil and its metabolites in human serum by high performance liquid chromatography

The antihypertensive agent urapidil (Ebrantil Byk-Gulden, Konstanz) can be reliably quantitated with three metabolites in human serum using high performance liquid chromatography. Serum is alkalinized and extracted with ethyl acetate. The organic phase is back-extracted with diluted acid. An aliquot is sampled automatically and chromatographed in an optimized combination of mobile and stationary phase. UV-detection at 273 nm allows a quantitation limit of 5 ng/ml for all analytes. Precise handling of exact volumes facilitates external calibration. The coefficient of variation for spiked samples is less than 5% within and less than 7% between studies. Application of the method to experimental and clinical pharmacokinetic studies of urapidil is illustrated.