血浆蛋白分子在单壁碳纳米管无纺膜表面吸附行为的研究

近年来,碳纳米管的独特表面拓扑结构、化学组成和优异的物理性能已经吸引了众多领域的研究兴趣,以生物医学应用为目标的探索性研究正在迅速形成一个新的方向。我们以血液接触环境下的应用为目标,通过扫描电镜观察、表面元素分析、以及利用酶联免疫分析技术,系统研究了与凝血过程密切相关的纤维蛋白原、白蛋白、免疫球蛋白以及新鲜血浆在单壁碳纳米管无纺膜(SWNT膜)表面的吸附行为。实验结果显示,单壁碳纳米管无纺膜对血浆中的纤维蛋白原分子具有强烈的倾向性吸附,对免疫球蛋白也显示出一定的吸附性,但是,对白蛋白分子却几乎不吸附。血浆蛋白分子在SWNT膜表面的吸附行为与其在其它碳材料表面和其它大多数生物材料表面的吸附行为显著不同。SWNT膜对血浆蛋白分子的独特吸附作用有可能对后续的血液细胞响应产生重要影响。     

用SPR生物传感器研究纤维蛋白原在生物医用材料表面？…

材料表面对血浆蛋白的吸附特性，是研究和评价生物医用材料血液相容性的重要依据。本文用自制的表面等离激元（ＳＰＲ）传感器，测量了金膜、磷脂ＤＳＰＣ膜、成都科大Ⅱ型聚氨酯、Ｐｅｌｌｅｔｈａｎｅ２３６３－５５Ｄ）聚氨酯及有机玻璃膜表面对纤维蛋白原的动态吸附特性，在纤维蛋白原溶液浓度为５ｍｇ／ｍｌ的相同条件下，磷脂ＤＳＰＣ膜表面吸附纤维蛋白原的速度最低，饱和吸附浓度也最小（表面浓度为１ｎｇ／ｍｍ＾２）。其次     

等离子体表面改性涤纶材料的血浆蛋白吸附与血小板黏附行为研究

采用等离子体表面接枝改性技术在涤纶 (聚对苯二甲酸乙二醇酯 ,PET)材料表面接枝不同分子量的聚乙二醇 (PEG) ,从表面能与界面自由能的角度分析了血浆蛋白 (纤维蛋白原和白蛋白 )在材料表面的竞争吸附关系 ,结果表明接枝了 PEG长链分子的 PET材料具有优先吸附白蛋白的性质 ,其中接枝 PEG6 0 0 0的 PET优先吸附倾向最明显。预接触白蛋白和纤维蛋白原的 PET材料表面的血小板黏附实验表明 :吸附白蛋白的表面能够显著抑制血小板的黏附和聚集 ,表现出好的血液相容性 ,而吸附了纤维蛋白原的材料表面具有降低血液相容性的性质。     

表面修饰密度对PEG化纳米颗粒与磷脂膜作用影响的动力学模拟

目的 初步研究聚乙二醇（PEG）分子表面修饰密度对于纳米颗粒和磷脂膜作用的影响.方法 应用粗粒度分子动力学计算方法,研究表面PEG修饰的纳米颗粒与细胞膜作用的动态过程.结果 通过对作用过程中PEG结构和能量的变化以及细胞膜构象的变化等参数的分析发现,修饰的PEG分子增大了颗粒的尺寸,并改善了刚性颗粒的表面弹性;PEG表面修饰能够抑制刚性纳米颗粒吸附所造成的磷脂膜弯曲形变,这种抑制作用与颗粒表面修饰的PEG分子密度相关.结论 PEG表面修饰能够抑制刚性颗粒吸附时所造成的磷脂膜的弯曲形变,此关系依赖于PEG表面修饰密度的变化;其所抑制膜弯曲的作用机理有可能限制细胞对纳米颗粒的有效吞噬,从而延长纳米载体在体内的循环时间.     

碳素材料表面吸附蛋白的红外光谱分析

为探讨类金刚石薄膜（DLC）、金刚石薄膜（DF）和石墨不同血浆蛋白吸附特性的内在原因，对人血白蛋白（HSA）和纤维蛋白原（HFG）在三种碳素材料表面吸附前后进行了红外光谱分析。结果显示：通过氨基，在HSA与DLC、HFG与DF、HFG与石墨之间形成了氢键，从而有力地支持并合理地解释了DLC具有较高的HSA吸附活性、DF和石墨则优先吸附HFG的前期研究结论。     

聚醚型聚氨酯材料表面纤维蛋白原的吸附性研究

本文采用放射性同位素标记的方法研究了嵌段聚醚型聚氨酯在纯纤维蛋白原溶液中和稀释血浆中的表面纤维蛋白原吸附性规律，考察了聚醚型聚氨酯的特性粘数及溶液体系中的ＮａＣｌ浓度对材料表面纤维蛋白原吸附性的影响，结果表明，随着聚合物特性粘数的增大，材料表面的纤维蛋白原吸附量呈降低的趋势；溶液体系中盐浓度的降低导致纤维蛋白原凝固性增强，在纯纤维蛋白原溶液中，材料表面纤维蛋白原的吸附量相应增多，而在稀释血浆中，纤维蛋白原的吸附量相应减少，在达到最低值后又有上升的趋向，表明纤维蛋白原在材料表面的吸附还受血浆中其它大分子的影响。     

血浆蛋白在聚苯乙烯—g—（二八烷聚氧乙烯）表面吸…

利用放射碘同位素标记技术研究了三种主要血浆蛋白质（白蛋白、免疫球蛋白或纤维蛋白原）在聚苯乙烯－ｇ－（十八烷聚氧乙烯）接枝共聚物表面的吸附动力学、等温吸附及竞争吸附。表面蛋白质吸附量和表面的ＳＰＥＯ含量非单调关系，与ＳＰＥＯ侧链的疏水末端基效应密切相关。二元蛋白质竞争吸附结果表明这三种蛋白质的相对竞争吸附能力为纤维蛋白原最大，免疫球蛋白次之，白蛋白最小。     

血浆蛋白质在聚苯乙烯-g-(十八烷聚氧乙烯)表面吸附的研究

利用放射碘同位素标记技术研究了三种主要血浆蛋白质（白蛋白、免疫球蛋白或纤维蛋白原）在聚苯乙烯－ｇ－（十八烷聚氧乙烯）接枝共聚物表面的吸附动力学、等温吸附及竞争吸附。表面蛋白质吸附量和表面ＳＰＥＯ含量非单调关系，与ＳＰＥＯ侧链的疏水末端基效应密切相关。二元蛋白质竞争吸附结果表明这三种蛋白质的相对竞争吸附能力为纤维蛋白原最大，免疫球蛋白次之，白蛋白最小。     

表面等离激元共振(SPR)生物传感器对聚氨酯材料血浆蛋白吸附的实时原位动态研究

应用实验室自行研制的自动扫描式表面激元共振(SPR)生物传感器对三种聚氨酯材料进行了血液蛋白质吸附实验，以传感片上的金膜作为对照材料。同时应用原子力显微镜对金膜和聚氨酯材料的超微结构与材料表面上所吸附的蛋白质进行了表征。实验结果显示，四种材料对纤维蛋白原和IgG的吸附量顺序均为：金膜＞H50—0＞H50—50＞H50—100。T—检验结果表明，金膜对纤维蛋白原和IgG吸附量与三种聚氨酯材料均有显著差别。该结果表明聚氨酯材料的血液相容性明显好于金膜对照材料。     

用SPR生物传感器研究纤维蛋白原在生物医用材料表面的吸附

材料表面对血浆蛋白的吸附特性 ,是研究和评价生物医用材料血液相容性的重要依据。本文用自制的表面等离激元 (SPR)传感器 ,测量了金膜、磷脂DSPC膜、成都科大Ⅱ型聚氨酯、Pellethane2 36 3 55D聚氨酯及有机玻璃膜表面对纤维蛋白原的动态吸附特性 ,在纤维蛋白原溶液浓度为5mg/ml的相同条件下 ,磷脂DSPC膜表面吸附纤维蛋白原的速度最低 ,饱和吸附浓度也最小 (表面浓度为 1ng/mm2 )。其次是裸金膜 (表面浓度为 3.5ng/mm2 ) ,再其次是成都科大Ⅱ型聚氨酯膜 (表面浓度为 3.8ng/mm2 )和Pellethane 2 36 3 55D聚氨酯 (表面浓度为 4 .3ng/mm2 ) ,吸附速度和吸附量最高的是有机玻璃膜 (表面浓度为 4 .5ng/mm2 )。结果表明 ,材料表面对纤维蛋白原的吸附动力学特性 ,与材料的血液相容性密切相关。表面等离激元技术与本文采用的在金膜上铺展高分子材料的离心铺膜法和LB技术等样品制备技术相结合 ,为生物材料表面对蛋白质吸附特性的实时、动态、原位研究提供了一种新的高灵敏度的方法 ,并可能发展成为一种材料生物相容性的测试和评价的新方法。     

Reduction of fibrinogen adsorption on PEG-coated polystyrene surfaces.

Reduction of protein adsorption by coating surfaces with polyethylene glycol (PEG) is well documented. The present work has four goals related to these previous studies: first, to develop chemistry providing densely packed, covalently bound PEG on polystyrene (PS); second, to determine the ability of these modified surfaces to reject fibrinogen; third, to compare the protein-rejecting ability of branched and linear PEGs; and fourth, to examine the utility of an ELISA-type procedure for measuring protein adsorption. It was found that PEG-epoxide could be readily coupled to amine groups of poly(ethylene imine) (PEI), which had been preadsorbed onto an oxidized PS surface. The PEG groups on branched PEGs appear to act as an excluded volume to repel proteins, similar to arguments previously raised for linear PEGs. The results of protein adsorption studies showed that fibrinogen adsorption is significantly reduced by coating polystyrene with either linear or branched PEGs of 1500 to 20,000 in molecular weight. The ELISA technique was found to be equivalent in sensitivity to radiolabeled fibrinogen for estimating adsorption levels. It is expected that PEG-coated PS will have much utility in a variety of biomedical applications.     

Interaction Between Polymer Chains Covalently Fixed to Single‐Walled Carbon Nanotubes

Summary: A single‐walled carbon nanotube (SWNT), which had been oxidized with a mixture of nitric acid and sulfuric acid to afford polar groups at its ends, was incubated with an azo‐type macroinitiator carrying dextran (DEX), poly(ethylene glycol) (PEG) or poly(N‐vinylpyrrolidone) (PVPy) chains at 70 °C. Similarly, the oxidized SWNT was incubated with 2,2′‐azoisobutyronitrile and acrylic acid (HAA) or N‐vinylpyrrolidone at 70 °C. Due to the large radical trapping ability of SWNT, the polymer chains corresponding to the cloven macroinitiator (PEG, DEX or PVPy) and the propagating polymer chains (poly(acrylic acid) (HPAA) or PVPy) were covalently fixed to the surface of the SWNTs. The hydrophilic polymer‐modified SWNTs could be stably dispersed in water. Furthermore, the SWNTs modified with PEG and DEX sedimented in the presence of free DEX and PEG, respectively, whereas there was no precipitation of the PEG‐ and DEX‐modified SWNTs in the presence of the same kind of free polymer. This seemed to be related to the phase separation phenomena in water soluble DEX and PEG systems induced by the repulsive interaction between PEG and DEX molecules. However, the mixture of two kinds of polymer‐modified SWNTs (PEG‐SWNT and DEX‐SWNT) did not show noticeable phase separation, probably due to steric hindrance for the efficient repulsive polymer‐polymer interaction by fixation to the gigantic SWNTs. Furthermore, upon mixing the dispersions of HPAA‐SWNT and PEG‐SWNT or PVPy‐SWNT, the turbidity of the dispersions gradually increased, while no increase in turbidity of the dispersion mixture was observed in the presence of dimethyl sulfoxide, indicating hydrogen bonding between the HPAA and PEG or PVPy chains on the surface of the SWNTs. The modification methods examined in this work would be promising to give various functions to SWNT.

Susceptible processes of radical trap on SWNT surface.     

Lysine-PEG-modified polyurethane as a fibrinolytic surface: Effect of PEG chain length on protein interactions,platelet interactions and clot lysis

Fibrinolytic polyurethane surfaces were prepared by conjugating lysine to the distal terminus of surface-grafted poly(ethylene glycol) (PEG). Conjugation was through the α-amino group leaving the ε-amino group free. Lysine in this form is expected to adsorb both plasminogen and t-PA specifically from blood. It was shown in previous work that the PEG spacer, while effectively resisting nonspecific protein adsorption, was a deterrent to the specific binding of plasminogen. In the present work, the effects of PEG spacer chain length on the balance of nonspecific and specific protein binding were investigated. PEG–lysine (PEG-Lys) surfaces were prepared using PEGs of different molecular weight (PEG300 and PEG1000). The lysine-derivatized surfaces with either PEG300 or PEG1000 as spacer showed good resistance to fibrinogen in buffer. The PEG300-Lys surface adsorbed plasminogen from plasma more rapidly than the PEG1000-Lys surface. The PEG300-Lys was also more effective in lysing fibrin formed on the surface. These results suggest that the optimum spacer length for protein resistance and plasminogen binding is relatively short. Immunoblots of proteins eluted after plasma contact confirmed that the PEG–lysine surface adsorbed plasminogen while resisting most of the other plasma proteins. The hemocompatibility of the optimized PEG–lysine surface was further assessed in whole blood experiments in which fibrinogen adsorption and platelet adhesion were measured simultaneously. Platelet adhesion was shown to be strongly correlated with fibrinogen adsorption. Platelet adhesion was very low on the PEG-containing surfaces and neither surface-bound lysine nor adsorbed plasminogen promoted platelet adhesion.     

Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer

A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 microg/cm2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.     

Modification of Polyurethane with Polyethylene Glycol–Corn Trypsin Inhibitor for Inhibition of Factor Xlla in Blood Contact

Abstract

In previous work using gold as a model substrate, we showed that modification of surfaces with poly(ethylene glycol) (PEG) and corn trypsin inhibitor (CTI) rendered them protein resistant and inhibitory against activated factor XII. Sequential attachment of PEG followed by CTI gave superior performance compared to direct attachment of a preformed PEG-CTI conjugate. In the present work, a sequential method was used to attach PEG and CTI to a polyurethane (PU) substrate to develop a material with applicability for blood-contacting medical devices. Controls included surfaces modified only with PEG and only with CTI. Surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy. The surface density of CTI was in the range of a monolayer and was higher on the PU substrate than on gold reported previously. Biointeractions were investigated by measuring fibrinogen adsorption from buffer and plasma, factor XIIa inhibition and plasma clotting time. Both the PU–PEG surfaces and the PU–PEG–CTI surfaces showed low fibrinogen adsorption from buffer and plasma, indicating that PEG retained its protein resistance when conjugated to CTI. Although the CTI density was lower on PU–PEG–CTI than on PU modified only with CTI, PU–PEG–CTI exhibited greater factor XIIa inhibition and a longer plasma clotting time, suggesting that PEG facilitates the interaction of CTI with factor XIIa. Thus sequential attachment of PEG and CTI may be a useful approach to improve the thromboresistance of PU surfaces.     

Molecular and supramolecular structure of adsorbed fibrinogen and adsorption isotherms of fibrinogen at quartz surfaces

The adsorption of fibrinogen to quartz surfaces was measured by ellipsometry, ELISA, and electron microscopy. The initial adsorption at low concentrations was diffusion rate limited as determined by the ELISA and by counting the number of adsorbed molecules at electron micrographs. From ellipsometry, ELISA, and electron microscopy measurements it was found that the surface concentration of adsorbed fibrinogen increased continuously over four decades in bulk concentration of fibrinogen. At a hydrophilic quartz surface a plateau level of the adsorption isotherm was found at a surface concentration of 0.1 pmol/cm2 where the adsorbed molecules had a mean intermolecular distance of 10 +/- 5 nm between neighbors. At higher surface concentrations the molecules were densely packed and formed a layer where single molecules could not be identified. Adsorbed fibrinogen showed different structure at hydrophobic and hydrophilic quartz surfaces. At a hydrophilic surface, the fibrinogen molecules appeared as a 46 nm nodose rod consisting of 6-7 nodes with a diameter of 4 nm. At a hydrophobic surface, the molecule appeared as a binodular or trinodular rod with a node diameter of 5-9 nm, connected with a thin filament to form a 40-nm rod. Adsorption from higher concentrations of fibrinogen in solution resulted in adsorbed spheric structures with a diameter of 18-24 nm at the hydrophobic surface and in end-to-end polymers at the hydrophilic quartz membrane.     

Protein adsorption to poly(ethylene oxide) surfaces.

Surfaces containing poly(ethylene oxide) (PEO) are interesting biomaterials because they exhibit low degrees of protein adsorption and cell adhesion. In this study different molecular weight PEO molecules were covalently attached to poly(ethylene terephthalate) (PET) films using cyanuric chloride chemistry. Prior to the PEO immobilization, amino groups were introduced onto the PET films by exposing them to an allylamine plasma glow discharge. The amino groups on the PET film were next activated with cyanuric chloride and then reacted with bis-amino PEO. The samples were characterized by scanning electron microscopy, water contact angle measurements, gravimetric analysis, and electron spectroscopy for chemical analysis (ESCA). The adsorption of 125I-labeled baboon fibrinogen and bovine serum albumin was studied from buffer solutions. Gravimetric analysis indicated that the films grafted with the low-molecular-weight PEO contained many more PEO molecules than the surfaces grafted with higher-molecular-weight PEO. The high-molecular-weight PEO surfaces, however, exhibited greater wettability (lower water contact angles) and less protein adsorption than the low-molecular-weight PEO surfaces. Adsorption of albumin and fibrinogen to the PEO surfaces decreased with increasing PEO molecular weight up to 3500. A further increase in molecular weight resulted in only slight decreases in protein adsorption. Protein adsorption studies as a function of buffer ionic strength suggest that there may be an ionic interaction between the protein and the allylamine surface. The trends in protein adsorption together with the water contact angle results and the gravimetric analysis suggest that a kind of "cooperative" water structuring around the larger PEO molecules may create an "excluded volume" of the hydrated polymer coils. This may be an important factor contributing to the observed low protein adsorption behavior.     

Measurement of fibrinogen adsorption from blood plasma using 125I-fibrinogen and a direct ELISA technique.

A direct enzyme-linked immunosorbent assay (ELISA), using a polyclonal anti-fibrinogen conjugated to horseradish peroxidase, was used to detect fibrinogen adsorption from blood plasma to ten different materials. Adsorption was also measured with [125I]-fibrinogen. The materials studied included glass, Biomer, Immulon I, and a series of hydroxyethylmethacrylate (HEMA) and ethylmethacrylate (EMA) co-polymers. For all the materials studied, the results from the ELISA technique closely paralleled those obtained using [125I]-fibrinogen. The cross-reactivity of the antibody with proteins other than fibrinogen was generally small. Both experimental methods detected the presence of a maximum in fibrinogen adsorption (as a function of the plasma dilution) to the more hydrophobic materials. For all but two HEMA/EMA co-polymers, a linear correlation between the ELISA and [125I]-fibronogen measurements was indicated by inspection of cross plots as well as by a statistical test.