PNIPAAm改性表面对蛋白质吸附的调控及其应用

根据不同领域的需要,控制蛋白质在材料表面的吸附是一个具有重要应用价值的课题。聚（N-异丙基丙烯酰胺）（PNIPAAm）改性表面能够响应外界温度变化从而改变其表面性质,这一特点为调控蛋白质的吸附提供了可能。近年来,研究者们应用多种表征方法考察了不同温度下蛋白质在PNIPAAm改性表面的吸附,并试图从分子水平上深入理解其吸附机制及影响因素。本文综述了近年来应用PNIPAAm改性表面对蛋白质吸附的研究及其最新进展。发现当PNIPAAm层厚度处于一定范围内时,PNIPAAm改性表面表现出对蛋白质吸附的温度敏感性,并可以利用这一性质将其应用于蛋白质纯化及分离和生物传感器等领域。而当PNIPAAm层厚度超过一个临界值时,PNIPAAm改性表面表现出良好的阻抗血浆蛋白质的性质,使其有望在血液相容性表面领域得到应用。最后,就PNIPAAm改性表面调控蛋白质吸附的未来发展方向简要地进行了展望。     

环状构象PEG改性表面及其优越的抗凝血性能

聚乙二醇(PEG)因其优异的抗蛋白质吸附能力成为抗凝血材料的首选.目前,多数研究都集中在PEG链长和接枝密度对蛋白质吸附的影响,鲜有关注PEG链构象影响的研究.本文利用硫金键在石英晶体微天平金片表面构建了两种不同分子量(MW=1000和MW=5000)的环状(SH-PEG-SH)和线型(m PEG-SH)构象的PEG改性表面,并研究了其抗纤维蛋白原吸附机理和抗凝血性能.X射线光电子能谱仪和原子力显微镜确定了不同表面的组成及其相结构.结果发现,环状构象的PEG表面相对于线型PEG构象能更加有效地抑制纤维蛋白原的吸附,同时具有更加优异的抗血小板和红细胞黏附性能.分析其蛋白质吸附机理发现,当PEG分子量较低时(MW=1000),其环状构象PEG表面抗纤维蛋白原吸附机理源于较高的表面覆盖率;当PEG分子量较高时(MW=5000),其抗纤维蛋白原吸附机理源于高黏弹性和高表面覆盖率共同作用的结果.本工作为构建抗凝血涂层提供了新的思路,并为制备高性能生物医用材料提供了理论基础.     

以PEG为间隔基固定赖氨酸制备血液相容的聚氨酯材料

通过多步表面改性方法制备了血液相容性好的聚氨酯材料. 以PEG为间隔基将ε-赖氨酸通过Schiff碱反应和进一步的还原反应连接于聚氨酯表面. 该表面的水接触角和XPS结果表明, PEG和ε-赖氨酸成功接枝. 用蛋白质吸附和血栓溶解实验对材料的血液相容性进行了研究. 蛋白质吸附结果表明, 相对于改性前的聚氨酯, ε-赖氨酸改性后的表面能减少纤维蛋白原的吸附量近80%. 血栓溶解测试结果显示, ε-赖氨酸改性后的表面能够在13 min内使初生的血栓溶解. 这些结果证实, 改性后的表面不仅能抑制非特异性蛋白质的吸附, 而且在测试条件下能溶解初生的血栓.     

水解可控功能切换型聚合刷的构筑及表面性能

采用表面受限光接枝技术在玻璃表面构筑末端酯键可水解的羧酸甜菜碱酯阳离子聚合物刷(PCBMA-1C2),通过调节氨水浓度控制羧酸甜菜碱酯末端酯键的水解程度,实现表面聚合物刷中季铵阳离子和羧酸甜菜碱两性离子基团分配比例的改变.通过X射线光电子能谱(XPS)和接触角测试表征了改性表面的化学结构和亲/疏水性能,通过蛋白质吸附和血小板黏附实验研究了玻璃表面改性前后及水解前后电荷性质对生物分子相互作用的影响.结果发现,当氨水浓度为0.1,0.2,0.3和0.4 mol/L时,聚合物刷PCBMA-1C2改性表面羧酸甜菜碱酯末端酯键的水解率分别为6%,43%,56%和~100%,随着水解程度的增大,改性表面牛血清白蛋白(BSA)的吸附量依次降低了3%,76%,93%,96%,纤维蛋白原(Fg)吸附量则依次降低了11%,45%,90%和96%;当水解率50%时,改性表面表现出优异的抗蛋白质吸附和血小板黏附性能.     

纤维蛋白原类淀粉样聚集膜的制备及其性能研究

利用血液中含量丰富的糖蛋白—纤维蛋白原,通过纤维蛋白原与还原剂三(2-羧乙基)磷盐酸盐(TCEP)反应,制备了基于纤维蛋白原类淀粉样聚集的纳米薄膜.利用荧光光谱、远紫外-圆二色谱等光学表征手段,探究了纤维蛋白原与TCEP反应的动力学过程及其二级结构的变化;通过透射电子显微镜对薄膜结构进行了表征;用原子力显微镜和光学椭偏仪探讨了反应时间、纤维蛋白原浓度、TCEP溶液pH值以及浓度对薄膜厚度的影响,通过原子力显微镜表征了其在不同溶剂中浸泡的稳定性;最后通过薄膜的血小板吸附实验和对蛋白质的吸附实验,表征了其抗污能力.实验结果表明:通过反应条件的控制,实现了对薄膜厚度的可控,且薄膜在不同的环境中表现出优异的稳定性;薄膜表现出了对血小板、蛋白质、生物体液(如胎牛血清、牛奶等)等非特异性吸附的良好抵抗能力,有望作为新一代生物基抗污材料运用于表/界面改性领域.     

pH响应性表面对蛋白质吸附的调控

通过表面引发原子转移自由基聚合在固定了引发剂的硅表面接枝了聚甲基丙烯酸叔丁酯(PtBMA),而后通过水解得到聚甲基丙烯酸(PMAA)聚合物刷.通过X射线光电子能谱、椭圆偏振仪和水接触角测试证明了接枝改性的成功.研究发现PMAA改性表面的浸润性和对蛋白质的吸附行为都具有一定的pH响应性.在较低pH值时改性表面相对疏水,随...     

糖化温敏凝胶的制备及其与HepG2细胞相互作用研究

利用自由基聚合法合成了半乳糖糖化温敏凝胶(P(NIPAAm-co-GAC))和壳聚糖糖化温敏凝胶(P(NIPAAm-co-CSA)),对其温度响应性和溶胀性能进行了研究,结果表明,两种糖化温敏凝胶在水中和细胞培养基中均显示较好的温度响应性,以及比聚(N-异丙基丙烯酰胺)温敏凝胶(PNIPAAm)更好的溶胀性能.进一步研究人肝肿瘤细胞(HepG2)在凝胶表面的细胞行为发现,HepG2在P(NIPAAm-co-GAC)、PNIPAAm凝胶表面吸附量及活性较高,表现出良好的生长趋势,而在P(NIPAAm-co-CSA)凝胶表面吸附量和活性很低,其增殖受到抑制;通过降低环境温度,能使培养在P(NIPAAm-co-GAC)和PNIPAAm凝胶表面的HepG2细胞发生自动脱附,避免了酶解法对细胞功能造成的损伤,并且细胞片层比单个细胞表现出更快的脱附速率;研究细胞转载行为表明,通过温度诱导得到的细胞片层,其生物活性远远大于通过酶解法得到的细胞的生物活性.     

聚苯乙烯-g-聚氧乙烯接枝共聚物表面阻抗蛋白质吸附的研究

利用放射性碘标记技术研究了血浆蛋白质-缓冲溶液体系在聚苯乙烯-g-聚氧乙烯接枝共聚物表面的等温吸附和吸附动力学。材料表面蛋白质等温吸附量或平衡吸附量随表面PEO含量增加而下降;吸附量最低值小于0.25μg,cm^-2;同时探讨了材料表面”梳状“结构对材料表面PEO侧链阻抗蛋白质性能的影响。     

MCM-41分子筛的改性、表面结构与吸附性质的研究

用三甲基氯硅烷、二甲基二氯硅烷、甲基三氯硅烷、四氯化硅与甲基碘改性高硅MCM-41中孔分子筛。XRD,^2^9SiMASNMR,^1^3CMASNMR,以及N~2、水与环己烷吸附的表征,显示改性不同程度地改变了分子筛的表面组成与结构,减小孔径,增加孔壁厚度,因而影响吸附行为,减小吸附容量。硅烷化减少了MCM-41的表面硅羟基含量,增加其疏水性。用CH~3I改性使孔径减小1.4nm,而硅羟基含量并未显著减少。硅烷化以及用CH~3I改性可提高MCM-41分子筛的热稳定性。SiCl~4的改性作用相对不明显。样品的水及环己烷吸附容量与其表面硅羟基含量呈现不同的线性关系,揭示高硅MCM-41分子筛表面吸附中心的本性。     

杂多酸的固载化研究(II)

研究了以多种方法化学改性的不同活性炭表面化学特性及十二硅钨酸（SiW_(12)）在相应表面的吸附. 首次提出，不同原料来源和改性的活性炭对SiW_(12)的吸附有不同的等温线形式和吸附力. 活性炭表面的羰基有利于SiW_(12)的吸附，而表面的羧基则是不利的．     

Poly(N-vinylpyrrolidone)-modified surfaces repel plasma protein adsorption

The present work aimed to study the interaction between plasma proteins and PVP-modified surfaces under more complex protein conditions.In the competitive adsorption of fibrinogen(Fg) and human serum albumin(HSA),the modified surfaces showed preferential adsorption of HSA.In 100%plasma,the amount of Fg adsorbed onto PVP-modified surfaces was as low as 10 ng/cm~2,suggesting the excellent protein resistance properties of the modified surfaces.In addition, immunoblots of proteins eluted from the modified surfaces after plasma contact confirmed that PVP-modified surfaces can repel most plasma proteins,especially proteins that play important roles in the process of blood coagulation.     

Adsorption of fibrinogen and lysozyme on silicon grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-initiated atom transfer radical polymerization

Surfaces based on grafted poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) "brushes" with a constant graft density of 0.39 chain/nm2 and chain length from 5 to 200 monomer units were prepared by surface-initiated atom transfer radical polymerization (ATRP) on silicon wafers. The chain length and layer thickness of the poly(MPC) grafts were varied via the ratio of MPC to sacrificial initiator. The surfaces were characterized by water contact angle, XPS, and AFM. The effect of poly(MPC) chain length on fibrinogen and lysozyme adsorption was studied in TBS buffer at pH 7.4. The adsorption of both proteins on the poly(MPC)-grafted surfaces was greatly reduced compared to the unmodified silicon. Adsorption decreased with increasing chain length of the poly(MPC) grafts. Grafts of chain length 200 (MW 59 000) gave adsorption levels of 7 and 2 ng/cm2, respectively, for fibrinogen and lysozyme at 1 mg/mL protein concentration, corresponding to reductions of greater than 98% compared to the unmodified silicon. Adsorption experiments using mixtures of the two proteins showed that the suppression of protein adsorption on the poly(MPC)-grafted surfaces was not strongly dependent on protein size or charge.     

Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study

Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities.     

Modification of polyurethane surface with an antithrombin-heparin complex for blood contact: influence of molecular weight of polyethylene oxide used as a linker/spacer

Polyurethane (PU) was modified using isocyanate chemistry to graft polyethylene oxide (PEO) of various molecular weights (range 300-4600). An antithrombin-heparin (ATH) covalent complex was subsequently attached to the free PEO chain ends, which had been functionalized with N-hydroxysuccinimide (NHS) groups. Surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS) to confirm the modifications. Adsorption of fibrinogen from buffer was found to decrease by ~80% for the PEO-modified surfaces compared to the unmodified PU. The surfaces with ATH attached to the distal chain end of the grafted PEO were equally protein resistant, and when the data were normalized to the ATH surface density, PEO in the lower MW range showed greater protein resistance. Western blots of proteins eluted from the surfaces after plasma contact confirmed these trends. The uptake of ATH on the PEO-modified surfaces was greatest for the PEO of lower MW (300 and 600), and antithrombin binding from plasma (an indicator of heparin anticoagulant activity) was highest for these same surfaces. The PEO-ATH- and PEO-modified surfaces also showed low platelet adhesion from flowing whole blood. It is concluded that for the PEO-ATH surfaces, PEO in the low MW range, specifically MW 600, may be optimal for achieving an appropriate balance between resistance to nonspecific protein adsorption and the ability to take up ATH and bind antithrombin in subsequent blood contact.     

Inhibition of protein adsorption and cell adhesion on PNIPAAm-grafted polyurethane surface: effect of graft molecular weight

In this work, the effect of molecular weight (MW) of surface grafted poly(N-isopropylacrylamide) (PNIPAAm) on protein adsorption and cell adhesion was investigated systematically. PNIPAAm-grafted polyurethane (PU) surfaces of varying graft MW were prepared via conventional radical polymerization. The MW was controlled by adjusting the monomer concentration. Fibrinogen (Fg) and human serum albumin (HSA) were selected as model proteins and their adsorption from phosphate-buffered saline (PBS, pH 7.4) and blood plasma at 37°C was measured using a radiolabeling method and immunoblot analysis respectively. It was found that in both media, as the MW increased, the adsorption of these two proteins decreased gradually reaching a plateau value at MW above 7.9×10(4). Compared to the unmodified PU, the surface grafted with PNIPAAm of MW 14.6×10(4) reduced the adsorption of Fg and HSA in PBS by 91% and 86%, respectively. Moreover, the surfaces with higher MW PNIPAAm showed minimal adhesion of L929 cells presumably due to the absence of cell-adhesive proteins on the surfaces.     

Fibrinolytic poly(dimethyl siloxane) surfaces

PDMS surfaces have been modified to confer both resistance to non-specific protein adsorption and clot lyzing properties. The properties and chemical compositions of the surfaces have been investigated using water contact angle measurements, ATR FT-IR spectroscopy, and XPS. The ability of the PEG component to suppress non-specific protein adsorption was assessed by measurement of radiolabeled fibrinogen uptake from buffer. The adsorption of plasminogen from human plasma to the various surfaces was studied. In vitro experiments demonstrated that lysine-immobilized surfaces with free epsilon-amino groups were able to dissolve fibrin clots, following exposure to plasma and tissue plasminogen activator. [Figure: see text].     

Protein resistant polyurethane surfaces by chemical grafting of PEO: amino-terminated PEO as grafting reagent

The objective of this work was to gain a better understanding of the mechanism of resistance to protein adsorption of surfaces grafted with poly(ethylene oxide) (PEO). A polyurethane-urea was used as a substrate to which PEO was grafted. Grafting was carried out by introducing isocyanate groups into the surface followed by reaction with amino-terminated PEO. Surfaces grafted with PEO of various chain lengths (PUU-NPEO) were prepared and characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). XPS data indicated higher graft densities on the PUU-NPEO surfaces than on analogous surfaces prepared using hydroxy-PEO (PUU-OPEO) as reported previously [J.G. Archambault, J.L. Brash, Colloids Surf. B: Biointerf. 33 (2004) 111-120]. Protein adsorption experiments using radiolabeled myoglobin, concanavalin A, albumin, fibrinogen and ferritin as single proteins in buffer showed that adsorption was reduced on the PEO-grafted surfaces by up to 95% compared to the control. Adsorption decreased with increasing PEO chain length and reached a minimum at a PEO MW of 2000. Adsorption levels on surfaces with 5000 and 2000 MW grafts were similar. There was no clear effect of protein size on resistance to protein adsorption. Adsorption on the PUU-NPEO surfaces was significantly lower than on the corresponding PUU-OPEO surfaces, again suggesting higher graft densities on the former. Adsorption of fibrinogen from plasma was also greatly reduced on the grafted surfaces. From analysis (SDS-PAGE, immunoblotting) of the proteins eluted after plasma exposure, it was found that the grafted surfaces and the unmodified substrate adsorbed the same proteins in roughly the same proportions, suggesting that adsorption to the PEO surfaces occurs on patches of bare substrate. The PEO grafts did not apparently cause differential access to the substrate based on protein size.     

Bioadhesive control of plasma proteins and blood cells from umbilical cord blood onto the interface grafted with zwitterionic polymer brushes

In this work, bioadhesive behavior of plasma proteins and blood cells from umbilical cord blood (UCB) onto zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymer brushes was studied. The surface coverage of polySBMA brushes on a hydrophobic polystyrene (PS) well plate with surface grafting weights ranging from 0.02 mg/cm(2) to 0.69 mg/cm(2) can be effectively controlled using the ozone pretreatment and thermal-induced radical graft-polymerization. The chemical composition, grafting structure, surface hydrophilicity, and hydration capability of prepared polySBMA brushes were determined to illustrate the correlations between grafting properties and blood compatibility of zwitterionic-grafted surfaces in contact with human UCB. The protein adsorption of fibrinogen in single-protein solutions and at complex medium of 100% UCB plasma onto different polySBMA brushes with different grafting coverage was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The grafting density of the zwitterionic brushes greatly affects the PS surface, thus controlling the adsorption of fibrinogen, the adhesion of platelets, and the preservation of hematopoietic stem and progenitor cells (HSPCs) in UCB. The results showed that PS surfaces grafted with polySBMA brushes possess controllable hydration properties through the binding of water molecules, regulating the bioadhesive and bioinert characteristics of plasma proteins and blood platelets in UCB. Interestingly, it was found that the polySBMA brushes with an optimized grafting weight of approximately 0.1 mg/cm(2) at physiologic temperatures show significant hydrated chain flexibility and balanced hydrophilicity to provide the best preservation capacity for HSPCs stored in 100% UCB solution for 2 weeks. This work suggests that, through controlling grafting structures, the hemocompatible nature of grafted zwitterionic polymer brushes makes them well suited to the molecular design of regulated bioadhesive interfaces for use in the preservation of HSPCs from human UCB.     

Colloid probe AFM investigation of interactions between fibrinogen and PEG-like plasma polymer surfaces

Interaction forces between surfaces designed to be protein resistant and fibrinogen (Fg) were investigated in phosphate-buffered saline with colloid probe atomic force microscopy. The surfaces of the silica probes were coated with a layer of fibrinogen molecules by adsorption from the buffer. The technique of low-power, pulsed AC plasma polymerization was used to make poly(ethylene glycol) (PEG)-like coatings on poly(ethylene teraphthalate) by using diethylene glycol vinyl ether as the monomer gas. The degree of PEG-like nature of the films was controlled by use of a different effective plasma power in the chamber for each coating, ranging from 0.6 to 3.6 W. This produced a series of thin films with a different number of ether carbons, as assessed by X-ray photoelectron spectroscopy. The interaction force measurements are discussed in relation to trends observed in the reduction of fibrinogen adsorption, as determined quantitatively by (125)I radio-labeling. The plasma polymer coatings with the greatest protein-repelling properties were the most PEG-like in nature and showed the strongest repulsion in interaction force measurements with the fibrinogen-coated probe. Once forced into contact, all the surfaces showed increased adhesion with the protein layer on the probe, and the strength and extension length of adhesion was dependent on both the applied load and the plasma polymer surface chemistry. When the medium was changed from buffer to water, the adhesion after contact was eliminated and only appeared at much higher loads. This indicates that the structure of the fibrinogen molecules on the probe is changed from an extended conformation in buffer to a flat conformation in water, with the former state allowing for stronger interaction with the polymer chains on the surface. These experiments underline the utility of aqueous surface force measurements toward understanding protein-surface interactions, and developing nonfouling surfaces that confer a steric barrier against protein adsorption.     

Protein-resistant and fibrinolytic polyurethane surfaces

Surfaces with resistance to non-specific protein adsorption and a high capacity to bind plasminogen from plasma are developed for application as fibrinolytic surfaces in blood contact. A new method is reported for grafting poly(OEGMA-co-HEMA) copolymers on polyurethane surfaces. The OEGMA provides effective protein resistance due to the PEG side chains and the HEMA provides a high density of OH groups for attachment of lysine. Adsorption of fibrinogen from buffer and plasma to these surfaces is low, indicating significant protein resistance. Plasminogen binding from plasma is high, and clot dissolution on surfaces where plasminogen adsorbed from plasma is converted to plasmin is rapid.