磷脂膜色谱及其在药物跨膜转运评价中的应用

磷脂膜色谱是固态基质上的有序磷脂分子单层体系采用色谱学方法仿真药物与细胞膜相互作用过程,可用来评价药物的细胞膜渗透性和活性。硅胶表面上的磷脂单分子层模拟了单层细胞膜,因此药物的磷脂膜色谱保留行为可用于预测药物与细胞膜的相互作用。目前考察药物跨膜转运的模型主要有正辛醇/水系统、脂质体/水系统、反相色谱（ODS）以及磷脂膜色谱。与前述3种系统比较,磷脂膜色谱除了具有高效、简便等特点外,同时能模拟药物与生物膜之间疏水作用力以外的其他作用力,因此对磷脂膜色谱的研究也越来越深入。由于药物细胞膜渗透性对其有效性和安全性起着关键作用,因此磷脂膜色谱在新药研发早期阶段的介入可以有效地降低后期候选药物的淘汰率,提高新药的研发效率。该文就磷脂膜色谱的研究及在药物跨膜转运评价中的应用进行了综述。     

髓鞘碱性蛋白对细胞膜脂质分子DPPC、DPPE单层膜影响机理研究

本文通过Langmuir单层膜的表面压力-平均分子面积(π-A)曲线的测定与分析,分别对髓鞘碱性蛋白(MBP)与细胞膜中不同头部基团脂质分子二棕榈酰基磷脂胆碱(DPPC)和二棕榈酰基磷脂酰乙醇胺(DPPE)在空气/液体界面上的相互作用过程进行了系统研究.实验结果表明:(1)当界面上脂质含量一定时,亚相中随着MBP浓度的增大,DPPC、DPPE单层膜的等温线向平均分子面积较大的方向移动;(2)在单层膜表面压力为10 mN/m时,一个MBP分子分别结合140±3个DPPC分子和100±3个DPPE分子,随着表面压力增大,当MBP分子分别与两种磷脂分子相互作用时,MBP插入到磷脂单层界面的个数逐渐减少;(3)随着蛋白质浓度的增加,脂分子形成的单层膜变得较为疏松,且MBP分子易于插入到分子头部较小的DPPE单层膜中;(4)蛋白质的存在使DPPC单层膜的表面压力逐渐减小,且蛋白质浓度越大表面压力降低越多,DPPC被MBP带入到亚相中越多;(5)对于DPPE单层膜,蛋白质通过与DPPE相互作用插入到界面膜中,引起表面压力增大,且蛋白质浓度越高,压力变化量越大.     

基于磷脂膜的界面相互作用研究

磷脂是一类非常重要的生物分子,它是细胞膜的主要组成部分,同时也在诸多生命活动（如细胞激活、代谢维持和激素分泌等）中发挥着不可替代的作用。磷脂种类繁多,且具有自组装能力强、生物相容性好、无细胞毒性、易于获得等一系列优点。作为一种最典型的界面材料,磷脂膜特殊的双层结构及出色的生物学性能引起了科研人员的广泛关注,其能够模拟生物膜结构,有助于研究界面上的分子特征及作用行为。此外,磷脂可被用作生物医学材料,改性磷脂以及磷脂与纳米颗粒的复合物在肿瘤成像技术、药物靶向递送系统等方面具有良好的发展前景,显著促进了新型生物材料的开发与进步。本文先归纳了磷脂的分类,并比较了磷脂在不同基底的吸附行为;之后重点分析了磷脂膜界面的选择性识别功能以及与多肽、酶、蛋白质等生物分子之间的相互作用;最后对基于磷脂膜的生物材料在生物传感、药物研究和成像技术中的应用作出了展望。     

含Schiff碱基囊泡双分子膜的聚集结构与相变

研究表面活性剂分子在水溶液中的聚集行为对模拟生物膜功能和研究分子间相互作用具有重要意义"'．用于形成囊泡双分子层的表面活性剂主要是类似天然磷脂的双烷基链两亲分子,单烷基链两亲分子在引人刚性基团时亦可形成双分子膜k'．含SChiff碱基两亲分子在水溶液中的聚集性质及间、尾链长度对SChiff碱基构象的影响已有报道"'．本文报道了这类分子的另一种重要成膜性质,即改变制备条件,可选择性地得到不同聚集结构和相变温度的双分子膜·实验中所用成膜分子为：CH。（CH。）。;OPh-N-CH－PhO（CH;）n;N"（CH。）。Br－（m-4;n-…     

人工细胞膜上天然酶与人工受体的分子间通讯

在集成的分子系统中,分子间的通讯联系是设计分子器件和分子机械的重要方式[1].在水相介质中自组装形成的脂质体可以作为分子间通讯的平台,各种分子可以依靠弱作用力,按照设计思路有组织、有计划地排布其上,构成一个功能化的超分子体,即纳米器件(nanodevice).在脂质体上模拟生物膜上发生的细胞信号转导,成为在分子和超分子水平上开发具有仿生特征的新型纳米器件的重要方法和手段,近年来成为关注的热点[2-6].     

血红蛋白与NO分子间相互作用的电化学表征

近年来 ,蛋白质的电化学研究引起了人们广泛兴趣[1～ 5] .利用自组装技术[4 ] 或将蛋白质分子固定于双层磷脂膜、水凝胶、表面活性剂膜及 Al2 O3膜上[6～ 9] 等可成为制备蛋白质膜的有效手段 .血红蛋白(Mr=6 50 0 0 )是生物体内一类重要的蛋白 ,由于对其结构已有较清楚的认识 [10 ] ,因而常被选作探讨生物大分子的电化学行为的模型分子 . NO是一种内皮细胞松弛因子、神经递质和免疫系统的媒介体 ,与生物体内的许多生理过程、疾病的产生与治疗有密切关系 [11～ 13] .因此研究蛋白与 NO之间的相互作用并寻求监测 NO的各种途径具有重要意义…     

不含蛋白质的功能泡囊膜及对酶的模拟

本篇综述了模拟生物膜的不含蛋白质的功能泡囊膜的建立过程,并介绍了这类功能泡囊膜所进行的模拟酶的泡囊催化反应。以磷脂或亲水亲油的表面活性剂分子所提供的双分子层泡囊结构为基质,分别在其不同的区域引入活性基团或分子,赋予了泡囊新的功能,由此可实现对一些反应的进程及产物构象的控制。利用这类功能泡囊膜,已成功地模拟了水解酶、还原酶、转氨酶、生物体内的碳单元传递反应等,并为模拟光合作用提供了一个很好的模式。     

生物分配色谱:高通量筛选药物膜通透性和活行

生物分配色谱是指在色谱系统中引入类生物膜结构,以色谱学方法仿真药物与生物膜的相互作用,现已成为评估药物膜通透性和活性的高通量筛选模型.根据最新进展并结合课题组研究内容,对其理论基础、分类及应用进行了评述,并对这一领域的发展和前景进行了展望.     

生物分配色谱:高通量筛选药物膜通透性和活行

生物分配色谱是指在色谱系统中引入类生物膜结构,以色谱学方法仿真药物与生物膜的相互作用,现已成为评估药物膜通透性和活性的高通量筛选模型。根据最新进展并结合课题组研究内容,对其理论基础、分类及应用进行了评述,并对这一领域的发展和前景进行了展望。     

生物分配色谱:高通量筛选药物膜通透性和活性

生物分配色谱是指在色谱系统中引入类生物膜结构,以色谱学方法仿真药物与生物膜的相互作用,现已成为评估药物膜通透性和活性的高通量筛选模型。根据最新进展并结合课题组研究内容,对其理论基础、分类及应用进行了评述,并对这一领域的发展和前景进行了展望。     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。     

The use of phospholipid modified column for the determination of lipophilic properties in high performance liquid chromatography

A new chromatographic stationary phase obtained by coating a reversed phase amide column with phosphatidylcholine based liposomes solution to yield a phospholipid modified column (PLM). The modification is achieved by the dynamic coating method which recycles the coating solution through the column in a closed loop for a period of 24 h. The chromatographic properties of the new column have changed significantly as compared to the original amide column due to the phospholipid coating. A good correlation was observed between n-octanol/water logP values and the logarithm of the retention factor obtained on the PLM column for a large number of solutes. In addition the PLM column was characterized using the linear solvation energy relationship (LSER). The values of the LSER system constants for the PLM column were calculated and were found to be very close to those of the n-octanol/water extraction system thus suggesting that the PLM column can be used for the estimation of n-octanol/water partition coefficient and serve as a possible alternative to the shake-flask method for lipophilicity determination. In addition, the results suggest that the PLM column can provide an alternative to other phospholipid-based column such as the IAM and the DPC columns.     

Structural Properties Governing Retention Mechanisms on Immobilized Artificial Membrane (IAM) HPLC Columns

The aims of this study were to investigate whether three commercially available immobilized artificial membrane (IAM) HPLC columns yield collinear data for neutral compounds, and whether IAM scales are distinct from the log P_oct (partition coefficient in the octanol/H₂O system) scale. With these objectives, the retention mechanisms on the IAM HPLC columns were analysed by linear solvation free‐energy relationships (LSERs). A set of 68 neutral model compounds with known solvatochromic parameters and log P_oct values was investigated, allowing a regular and broad exploration of property space. The resulting solvatochromic equations clearly indicate that the three IAM stationary phases retain small neutral solutes by a balance of intermolecular forces closely resembling those underlying partitioning in octanol/H₂O and retention on a reversed‐phase LC‐ABZ HPLC column. For all systems, the solute's size and hydrogen‐bond‐acceptor basicity are the two predominant factors, whereas dipolarity/polarisability and hydrogen‐bond‐donor acidity play only minor roles.     

Computer simulation of the distribution of hexane in a lipid bilayer: spatially resolved free energy, entropy, and enthalpy profiles

The partitioning behavior of small molecules in lipid bilayers is important in a variety of areas including membrane protein folding and pharmacology. However, the inhomogeneous nature of lipid bilayers on a nanometer length scale complicates experimental studies of membrane partitioning. To gain more insight in the partitioning of a small molecule into the lipid bilayer, we have carried out atomistic computer simulations of hexane in a dioleoyl phosphatidylcholine model membrane. We have been able to obtain spatially resolved free energy, entropy, enthalpy, and heat capacity profiles based on umbrella sampling calculations at three different temperatures. In agreement with experiment, hexane partitions preferentially to the center of the bilayer. This process is driven almost entirely by a favorable entropy change, consistent with the hydrophobic effect. In contrast, partitioning to the densest region of the acyl chains is dominated by a favorable enthalpy change with a small entropy change, which is consistent with the "nonclassical" hydrophobic effect or "bilayer" effect. We explain the features of the entropy and enthalpy profiles in terms of density and free volume in the system.     

Immobilised artificial membrane chromatography coupled with molecular probing. Mimetic system for studying lipid-calcium interactions in nutritional mixtures

Immobilised artificial membrane (IAM) chromatography was utilised to study the interactions of usual membrane probes with grafted phosphatidylcholine silica support, in relation to the presence of calcium ions introduced in the mobile phase as they are present in nutritional mixtures. IAM acts as a mimetic membrane of lipid emulsion globules, a major component of nutritional mixtures. The tested probes were 1,6-diphenyl-1,3,5-hexatriene (DPH), 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one or nile red (NR) and 2-(p-toluidinyl)naphtalene-6-sulfonate (TNS). For each probe, partition coefficients and thermodynamic parameters of transfer from the mobile phase to the IAM stationary phase have been measured. Our results suggested that the interactions of neutral probes (i.e. DPH and NR) with phosphatidylcholine are driven by hydrophobic forces. Addition of calcium chloride to the mobile phase slightly decreased the retention of these neutral probes and dramatically increased that of anionic TNS. Moreover, an enthalpy-entropy compensation study revealed that the mechanism of interaction between TNS and IAM is independent of the calcium concentration. Results argued for the existence of electrostatic repulsion forces exerted by IAM phase towards anionic TNS. Addition of calcium ions into the mobile phase led to the establishment of an ionic double layer at the zwitterionic stationary phase surface weakening the electrostatic barrier and increasing TNS retention. Consequently, it was demonstrated that IAM appears as a suitable model to get a better insight on the lipid-calcium interactions taking place in nutritional mixtures.     

Drug–Membrane Interaction on Immobilized Liposome Chromatography Compared to Immobilized Artificial Membrane (IAM), Liposome/Water,and Octan‐1‐ol/Water Systems

The objective of this study was to investigate drug–membrane interaction by immobilized liposome chromatography (ILC; expressed as lipophilicity index log K_s) and the comparison with lipophilicity indices obtained by liposome/H₂O, octan‐1‐ol/H₂O, and immobilized artificial membrane (IAM) systems. A set of structurally diverse monofunctional compounds and drugs (nonsteroidal anti‐inflammatory drugs and β‐blockers) were selected in this study. This set of solutes consists of basic or acidic functionalities which are positively or negatively charged at physiological pH 7.4. No correlation was found between log K_s from ILC and lipophilicity indices from any of the other membrane model systems for the whole set of compounds. For structurally related compounds, significant correlations could be established between log K_s from ILC and lipophilicity indices from IAM chromatography and octan‐1‐ol/H₂O. However, ILC and liposome/H₂O systems only yield parallel partitioning information for structurally related large molecules. For hydrophilic compounds, the balance between electrostatic and hydrophobic interactions dominating drug partitioning is different in these two systems.     

Quantitative structure-retention relationship studies using immobilized artificial membrane chromatography I: amended linear solvation energy relationships with the introduction of a molecular electronic factor

Linear solvation energy relationships (LSERs) amended by the introduction of a molecular electronic factor were employed to establish quantitative structure-retention relationships using immobilized artificial membrane (IAM) chromatography, in particular ionizable solutes. The chromatographic indices, log k(IAM), were determined by HPLC on an IAM.PC.DD2 column for 53 structurally diverse compounds, including neutral, acidic and basic compounds. Unlike neutral compounds, the IAM chromatographic retention of ionizable compounds was affected by their molecular charge state. When the mean net charge per molecule (delta) was introduced into the amended LSER as the sixth variable, the LSER regression coefficient was significantly improved for the test set including ionizable solutes. The delta coefficients of acidic and basic compounds were quite different indicating that the molecular electronic factor had a markedly different impact on the retention of acidic and basic compounds on IAM column. Ionization of acidic compounds containing a carboxylic group tended to impair their retention on IAM, while the ionization of basic compounds did not have such a marked effect. In addition, the extra-interaction with the polar head of phospholipids might cause a certain change in the retention of basic compounds. A comparison of calculated and experimental retention indices suggested that the semi-empirical LSER amended by the addition of a molecular electronic factor was able to reproduce adequately the experimental retention factors of the structurally diverse solutes investigated.     

Characterization of immobilized artificial membrane (IAM) and XTerra columns by means of chromatographic models

Immobilized artificial membranes (IAMs) prepared from phosphatidylcholine analogs are used as stationary phases in liquid chromatography systems to model drug partitioning between an aqueous phase (mobile phase) and a cell membrane (IAM column). Two different chromatographic models, which describe retention as a function of solute and column-mobile phase properties, have been applied to characterization of an IAM and two reversed phase C18 columns (Waters XTerra MSC18 and XTerra RP18) with acetonitrile-water mobile phases. The comparison of the results shows that the phosphatidylcholine group makes IAM column more polar than both XTerra columns, specially in terms of hydrogen-bond acceptor ability. XTerra RP18 is slightly more polar than XTerra MSC18 because of the presence of the embedded carbamate polar group.     

Retention Behavior of Neutral and Positively and Negatively Charged Solutes on an Immobilized‐Artificial‐Membrane (IAM) Stationary Phase

The retention behavior of neutral, positively charged, and negatively charged solutes on the IAM.PC.DD2 stationary phase was investigated and compared. A set of monofunctional compounds and complex drugs (steroids, nonsteroidal anti‐inflammatory drugs, and β‐blockers) were selected for this study, i.e., neutral solutes and solutes with acidic or basic functionalities which are positively charged or negatively charged at pH 7.0. The correlation between the retention factor log k_w at pH 7.0 on the IAM.PC.DD2 stationary phase and the partition coefficient log P_oct or the distribution coefficient log D_7.0 showed that the retention mechanism depends on the charge state and structural characteristics of the compounds. The neutrals were least retained on the IAM.PC.DD2 stationary phase, and positively charged solutes were more retained than negatively charged ones. This implies that the retention of the charged solutes is controlled not only by lipophilicity but also by the electrostatic interaction with the phospholipid, with which positively charged solutes interact more strongly than negatively charged ones.