不同硬段含量嵌段聚醚氨酯的合成、性能及莫膜上丙烯酰胺的接枝聚合

本文报告以4，4′—二苯甲烷二异氰酸酯、聚四氢呋喃—环氧丙烷共聚醚（Mn=2300），1，4—丁二醇为原料，用两步溶液聚合法合成了四种不同硬段含量的嵌段聚醚氨酯并测定了性能。四种聚醚氨酯的硬段含量分别为29％，46％，63％与78％。测定的性能有动态力学性能、物理—机械性能以及抗凝血性能。并用铈盐引发丙烯酰胺在这四种聚合物的膜上进行了接枝聚合。研究了硬段含量对聚醚氨酯的性能与接枝能力的影响。     

生物活性短肽RGD在PET表面接枝方法的研究

在高分子材料表面共价引入人工合成的精氨酰-甘氨酰-天冬氨酸三肽(Arg-Gly-Asp peptides，RGD)，以达到让内皮细胞与之特异结合并更加牢固的目的。实验用紫外辐照法将活性基团羧基(-COOH)接枝到聚对苯二甲酸乙二醇酯(Poly(ethylene terephtalate)，PET)膜的表面，将液相合成的RGD三肽耦合接枝到处理过的材料表面，光电子能谱对以羧基为活性基团的接肽反应结果进行分析，光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。内皮细胞生长实验结果表明，成功接枝的RGD序列对材料内皮细胞种植起到了促进作用。本实验成功运用紫外接枝与化学耦合，将生物活性短肽RGD接枝到膜表面，探索了一种新的接枝生物活性短肽的方法。     

聚砜中空纤维血液透析膜的制备与表征

为满足医学治疗的需要,提出一种以期用于血液透析的聚砜中空纤维膜制备方法。以聚砜(PSF)为膜材料,一缩二乙二醇(DEG)为致孔剂,聚乙烯吡咯烷酮(PVP-K30)为改性剂,N,N-二甲基乙酰胺(DMAC)为溶剂,应用非溶剂致相分离法(NIPS)制备中空纤维血液透析膜。采用扫描电镜、透析实验、接触角和孔隙率测试、拉伸测试等方法,表征不同质量分数的聚砜(17%、19%、21%、23%)对膜的微观结构、渗透性能、亲水性和机械性能的影响。利用肌酐、磷酸氢二钠和维生素B₁₂配制成模拟液,进行模拟血液透析实验,测试不同聚砜质量分数的透析膜对溶质的清除效果。对不同分子量(1 000、2 000、4 000、6 000、10 000、20 000)聚乙二醇(PEG)的水溶液进行溶质截留测试,计算得到透析膜的平均孔径和切割分子量。结果表明:当铸膜液中PSF的质量分数为21%,添加剂DEG和PVP的质量分数均为10%时,能够得到性能最优的透析膜;所得透析膜超滤系数达185.1 mL/m²·h·mmHg,对中小型分子的清除率达50%,断裂强度达5.22 MPa,膜表面孔径为5.00 nm,切割分子量为14 200。基于其良好的渗透性能和机械性能,该聚砜中空纤维膜有成为理想的血液透析膜材料的潜在可能。     

介入导管材料的表面紫外光接枝润滑改性

研究了用紫外光直接引发亲水性单体在聚氨酯 ( PU)表面进行接枝反应。以改善 PU表面的润滑性。实验测定了单体浓度对接枝密度、吸水率、摩擦系数的影响 ,对不同反应方法得到的结果进行了比较 ,找到了获得最佳润滑表面的条件 ,并对接枝聚合物的表面进行了扫描电镜 ( SEM)观察。实验结果表明 :接枝亲水性单体可大大地改善聚氨酯表面的润滑性     

聚乳酸-聚乙二醇共聚物/表面接枝复合纤维材料与成骨细胞的黏附、增殖及活性

背景:大量的研究表明,应用于骨组织工程的材料,如羟基磷灰石及高分子聚乳酸等,均对骨缺损的修复起到了一定的作用。但单一的材料在组织相容性及仿生学等特点诸多方面,无法满足骨组织生长的需要,将现有的单一材料进行改性再复合产生新的材料是骨组织工程发展的一个重要方向。目的:通过静电纺丝技术制备共混的聚乳酸-聚乙二醇共聚物/表面接枝羟基磷灰石复合纤维材料,探讨表面接枝羟基磷灰石的引入及其在材料中的含量对成骨细胞在新型材料黏附、增殖和细胞活性的影响。方法:通过调整静电纺丝溶液中聚乳酸-聚乙二醇共聚物和表面接枝羟基磷灰石的4个质量百分数比例,5%/0%,5%/5%,5%/10%,5%/15%,分别制备的4种新型材料(样品1,2,3,4)与小鼠成骨细胞共培养,在特定的时间点,通过细胞染色、碱性磷酸酶检测等实验方法,获得4种不同材料对与之共培养的成骨细胞的相关数据。通过对上述数据进行统计分析,评价新型材料对成骨细胞黏附、增殖能力及细胞活性的影响。结果与结论:在细胞黏附实验中,3组含有表面接枝羟基磷灰石成分的新型材料与不含的材料相比,细胞数量均增长明显,各组细胞增殖情况和碱性磷酸酶活性从高到低依次为样品3组样品4组样品2组样品1组(P0.05)。结果证实,聚乳酸-聚乙二醇共聚物中引入表面接枝羟基磷灰石后其生物活性得到提高,且新型材料中表面接枝羟基磷灰石含量可影响材料与成骨细胞相容性及细胞活性。     

腹膜表面超微结构的观察

目的：证实大鼠腹膜表面液体层的存在并探讨其可能组成部分。方法：取正常大鼠膜膜组织,分别以不同固定液固定：（1）2．5％戊二醛和2％多聚甲醛（对照组）;（2）2．5％戊二醛和0．5％,氯化十六烷吡啶（GAG组）;（3）2％O8O4（PH1组）;（4）4％OsO4（PH2组）;（5）3％鞣酸（鞣酸组）。结果对照组中可见腹膜间皮细胞表面的微绒毛,未见表面液体层。GAG组腹腊有面可见一层不连续的无定形结构,此层结构在PH1和PH2组中保存较好,厚度达到10μm。鞣酸组表面液体层及间皮细胞中见许多类板层小体。结论：研究证实：正常大鼠腹膜表面覆盖症一层膜样结构,它至少由磷脂和葡糖胺聚糖组成,该层在腹膜物质转运中可能有重要的屏障作用。     

001 聚氨酯表面接枝左旋-丙交酯以改善细胞粘附性

[英]/Hsu Shan-Hui… //Biomaterials. - 2000, 21(4).-359～367 聚氨酯具有良好的生物相容性和力学性能,在心血管材料及其它生物医用材料领域得到广泛应用,但其表面的细胞粘附性较差,本文研究了运用等离子体引发表面接枝方法改善聚氨酯表面的细胞粘附性.市售聚氨酯溶于四氢呋喃铸成膜,用氩等离子体处理后在空气中放置一定时间,浸入溶有左旋丙交酯的甲苯中,脱气封管在70℃反应5h,得到表面接枝左旋丙交酯的PU膜.接触角测量显示等离子体处理后水接触角由73°下降到36.,接枝使接触角略有上升,但仍比未处理聚氨酯及聚左旋乳酸表面亲水性好.只用等离子体处理而未进行表面接枝的聚氨酯膜接触角会逐渐升高,原因是表面亲水基团的重新分布.表面接枝后即无此现象.ESCA分析显示处理后表面基团发生变化,O/C比上升,有新的表面基团形成,且结构也不同与本体聚合的聚左旋乳酸.经过如此表面接枝处理的聚氨酯膜对成纤维细胞的粘附性较未处理聚氨酯膜有了成倍增长,也优于仅用等离子体处理的聚氨酯膜,对上皮细胞的粘附情形也类似.SEM观察显示粘附在接枝处理后的聚氨酯膜表面的细胞更为伸展.而血小板在接枝处理过的聚氨酯膜表面的粘附量及活性明显下降.总之,运用等离子体引发聚氨酯表面接枝左旋丙交酯获得了更好的血液相容性,具有潜在的广泛应用前景. (陈晓东摘朴东旭校)     

透析器复用过程中主要黏附物成分研究

本研究以复用的聚砜膜透析器为研究对象,对造成透析膜堵塞的黏附物质的主要成分作定量分析研究,并分析了不同复用次数的透析器膜表面残留成分的差异。结果显示:透析后用反渗水冲洗透析器,可去除膜表面黏附的大量蛋白质;用2%的次氯酸钠(NaClO)溶液浸泡使用后的透析器,可去除黏附物中的葡萄糖、胆固醇和甘油三脂;膜上大多数黏附物质随着透析次数的增加而缓慢增加。     

三嵌段高分子骨组织工程支架材料的表面多肽修饰与细胞粘附性研究

目的用甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸对三嵌段高分子骨组织工程支架材料进行表面修饰，并检测其细胞粘附性。方法利用异双官能交联剂将甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸多肽固定在聚丙交酯／乙交酯／天冬氨酸-聚乙二醇材料表面，并进行X线光电子分光镜检测和表面接触角测定；体外培养骨髓基质细胞，接种至表面修饰的材料上，测定细胞粘附力，并和未修饰材料对比。结果固定交联剂和多肽后X线光电子分光镜检测示硫元素的含量分别为0．3％和0．2％；硫元素的结合能是164eV和163．9eV；表面接触角为60．2±2．364度；细胞粘附力为（521．45±134．98）×10^-10牛顿。结论甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸能共价固定在聚丙交酯／乙交酯／天冬氨酸-聚乙二醇材料表面；多肽修饰后的材料能特异性的介导骨髓基质细胞粘附，增强其粘附力。     

一种检测血清心肌肌钙蛋白Ⅰ的生物传感器

目的：建立一种新的血清心肌肌钙蛋白Ⅰ(cTnI)定量检测法。方法：用亲和层析法提纯cTnI，免疫BALB／C鼠及新西兰兔，并用杂交瘤技术及膜渗滤亲和层析法，制备了特异性抗cTnI单克隆抗体和多克隆抗体。以葡萄球菌蛋白A作基底膜，特异性多克隆抗体作捕捉抗体，单抗9F5作第二抗体，制成了表面等离子体共振生物传感器。比较了直接法和夹心免疫法检测血清cTnI的性能。结果：夹心免疫法的最低检测限(0．8μg／L)是直接法的5倍(4μg／L)，检测范围为(0．8～20)μg／L，批内及批间精密度分别达3．8％～5．1％，6．3％～8．1％。用该夹心法及国外试剂盒分别检测40名健康献血队员和28例急性心肌梗死患者血清cTnI水平，两者符合率分别为97．5％和94．6％。结论：所建立的夹心免疫法操作简单，特异性强，灵敏度高，符合临床诊断要求。     

Biomolecular modification of p(AAm-co-EG/AA) IPNs supports osteoblast adhesion and phenotypic expression

Interpenetrating polymer networks (IPNs) were designed to resist materials fouling caused by non-specific protein adsorption, and indiscriminate cell or bacterial adhesion. These IPNs were thin adherent films ( ~ 20 nm) comprised of acrylamide (AAm), ethylene glycol (EG), and acrylic acid (AA) grafted to either silicon waters or quartz substrates via photoinitiated free radical polymerization. These networks were further modified to promote specific cell adhesion by tethering bioactive groups such as peptides that mimic cell-binding domains found on extracellular matrix molecules. As a specific example of biomolecular surface engineering, peptides from the cell-binding domain of bone sialoprotein were tethered to a p(AAm-co-EG/AA) IPN to control cell behavior at the surface. The networks were characterized by contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy to convey information on IPN wettability, thickness, and chemistry. The surface characterization data supported the theory that the PEG/AA layer formed an IPN with the underlying p(AAm) network, and after graft modification of this IPN with diamino PEG (PEG(NH₂)₂), the PEG(NH₂)₂ chains were enriched at the surface. Rat calvarial osteoblasts attached to Arg-Gly-Asp (RGD) modified IPNs at levels significantly greater than on clean quartz, Arg-Gly-Glu (RGE) modified, or the PEG(NH₂)₂ modified IPN, with or without serum in the media. Cells maintained in media containing 15% fetal bovine serum (FBS) proliferated, exhibited nodule formation, and generated sheets of mineralized extracellular matrix (ECM) with the addition on β-glycerophosphate to the media. Cell adhesion and mineralized ECM formation were specifically dependent on the peptide sequence present at the surface.     

Polyethylene glycol-grafted polystyrene particles

Densely pegylated particles that can serve as a model system for artificial cells were prepared by covalently grafting amino polyethylene glycol (PEG, molecular weight 3400 or 5000) onto carboxyl polystyrene particles (PS-COOH) using carbodiimide chemistry. PEG-modified particles (PS-PEG) were characterized by determination of the PEG surface concentration, zeta-potential, size, and morphology. Under optimized grafting conditions, a dense "brush-like" PEG layer was formed. A PEG surface concentration of approximately 60 pmol/cm2, corresponding with an average distance between grafted PEG chains of approximately 17 A can be realized. It was shown that grafting of PEG onto PS-COOH reduced the adsorption of proteins from human plasma (85 vol %) in phosphate-buffered saline up to 90%.     

Folic Acid Modified Poly(lactide‐co‐glycolide) Nanoparticles,Layer‐by‐Layer Surface Engineered for Targeted Delivery

The layer‐by‐layer (LbL) assembly technique was applied for the surface modification of biodegradable poly(lactide‐co‐glycolide) nanoparticles (NPs), employing poly(acrylic acid) (PAA), and polyethylenimine (PEI) as building blocks. Amino terminated poly(ethylene glycol) (PEG) and folate decorated PEG (PEG‐FA) were grafted onto the multilayers via condensation between carboxylic groups and amine groups from PEG or PEG‐FA. The LbL assembly and the covalent functionalization were monitored by means of ζ‐potential measurements and the quartz crystal microbalance with dissipation technique (QCM‐D). Protein adsorption after incubation of the NPs in culture medium containing optionally the serum proteins was investigated and related to cellular uptake. Experiments on cellular uptake showed that after PEGylation the uptake ratio of the NPs decreased significantly, but became three times larger when PEG‐FA was grafted on the NPs instead of the PEG.

     

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.     

Dual surface modification with PEG and corn trypsin inhibitor: effect of PEG:CTI ratio on protein resistance and anticoagulant properties

The objective of this study was to investigate the bioactivity and protein-resistant properties of dual functioning surfaces modified with PEG for protein resistance and corn trypsin inhibitor (CTI) for anticoagulant effect. Surfaces on gold substrate were prepared with varying ratios of free PEG to CTI-conjugated PEG. Two methods designated, respectively, "sequential" and "direct" were used. For sequential surfaces, PEG was first immobilized on gold and the surfaces were incubated with CTI at varying concentration. For direct surfaces, a PEG-CTI conjugate was synthesized and gold surfaces were modified using solutions of the conjugate of varying concentration. The CTI density on these surfaces was measured using radiolabeled CTI. Water contact angles were measured and the thickness of PEG-CTI layers was determined by ellipsometry. Fibrinogen adsorption from buffer and human plasma, and adsorption from binary solutions of fibrinogen and α-lactalbumin were investigated using radiolabeling methods. Bioactivity of the surfaces was evaluated via their effects on FXIIa inhibition and plasma clotting time. It was found that as the ratio of CTI-conjugated PEG to free PEG increased, bioactivity increased but protein resistance was relatively constant. It is concluded that on these surfaces conjugation of PEG to CTI does not greatly compromise the protein resistance of the PEG but results in improved interactions between the CTI and the "target" protein FXIIa. At the same CTI density, sequential surfaces were more effective in terms of inhibiting FXIIa and prolonging clotting time.     

Preparation of a PEG-grafted phospholipid Langmuir-Blodgett monolayer for blood-compatible material

In order to develop a versatile model for blood-compatible materials, we studied morphology and platelet adhesion of a dipalmitoyl-phosphatidylcholine (DPPC)/dipalmitoyl-phosphatidylethanolamine-polyethylene glycol (PEG lipid) mixed monolayer. This monolayer, which mimics the cell membrane structure, consists of two heterogeneous layers, that is, a PEG layer lying on top of a phospholipid monolayer. The DPPC/PEG lipid mixed monolayer was prepared using the Langmuir-Blodgett (LB) Technique. The monolayer was transferred onto a silanized glass substrate by the down-stroke mode, at a surface pressure of 25 mN/m. The transfer efficiency achieved unity at all times. The morphologies of PEG chains on the phospholipid monolayer in water, in a dried state, and in a hydrated state were evaluated using Pi-A isotherm, ellipsometry, and atomic force microscopy (AFM), respectively. When the concentration of PEG lipid was below 1 mol %, the PEG chains could cover the DPPC surface completely in water, but not in the dried state. On the other hand, the PEG chains could cover the phospholipid surface completely in a dried state, as well as in water, when the PEG lipid concentration was above 3 mol %. These PEG chains, showing a brush-type conformation in water, were highly packed and had a bulky structure at the surface in the dried state as well as in the hydrated state. A bulky and extended PEG layer, above 3 mol % concentration, was greatly effective in the prevention of platelet adhesion.     

Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes

Fabrication of functional tissue constructs from designed three-dimensional structures of cells using the layered method of cultured cell sheets could prove to be an attractive approach to tissue engineering. Rapid recovery of cell sheets is considered to be important as a basic technology for practical assembly of tissue-mimicking structures. To accelerate required culture substrate hydrophilic/hydrophobic functional changes according to the hydrated/dehydrated structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted with poly(ethylene glycol) (PEG) onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform infrared and electron spectroscopy for chemical analysis revealed that PIPAAm and PEG were successfully grafted to surfaces of porous membranes. PIPAAm-grafted porous membranes (PIPAAm-PM) were compared with porous membranes co-grafted with various amounts of PEG and PIPAAm (PIPAAm(PEG)-PM) for cell sheet detachment experiments. Approximately 35min incubation at 20 degrees C was required to completely detach cell sheets from PIPAAm-PM in a static condition, while only 19min to detach cell sheets from PIPAAm(PEG0.5%)-PM, which is co-grafted with PIPAAm and 0.5wt% of PEG. With porous membranes, water molecules were accessed by the PIPAAm molecules grafted on the surfaces from both underneath and peripheral to the attached cell sheet, resulting in more rapid hydration of grafted PIPAAm molecules and detachment of cell sheet than that for nonporous tissue culture polystyrene (TCPS) dish. With PIPAAm(PEG)-PMs, grafted PEG chains should accelerate the diffusion of water molecules to PIPAAm grafts, showing more rapid detachment of cell sheet compare to PIPAAm-PMs.     

Rapid and Efficient Assembly of Functional Silicone Surfaces Protected by PEG: Cell Adhesion to Peptide-Modified PDMS

While silicone elastomers generally have excellent biomaterials properties, their hydrophobicity can elicit undesired local biological responses through adsorption and denaturation of proteins. Surface-bound poly(ethylene glycol) (PEG) can ameliorate the situation by preventing contact between the external biology and the silicone elastomer. It is further possible to manipulate the biocompatibility of the surface by linking peptides, proteins or other biological entities to the PEG. Previous synthetic approaches to PEG-protected surfaces are compromised by issues of reproducibility. We describe two rapid and efficient approaches to silicone surface modification by PEG-linked adhesion peptides that overcome this problem: SiH groups are introduced throughout a silicone elastomer during elastomer synthesis or only at the surface after cure; then, in either case, protein-repellent PEG brushes at the surface are introduced by hydrosilylation to give surfaces that can be stored for extensive periods of time without degradation. Activation of the free alcohol with an NSC group followed by immediate conjugation to relevant biological molecules occurs in high yields, as shown for RGDS and GYRGDS. High surface grafting density of the peptides was demonstrated using radiolabeling techniques. Biological activity was demonstrated by a 5-fold increase in cell adhesion on the peptide-modified surfaces when compared to unmodified PDMS control surfaces.     

Mechanisms of transcellular transport of wheat germ agglutinin-functionalized polymeric nanoparticles in Caco-2 cells

Transcellular transport is essential for transmucosal and plasma-to-tissue drug delivery by nanoparticles, whereas its fundamental pathways have not been fully clarified. In this study, an in-depth investigation was conducted into the intracellular itinerary and the transcytosis pathway of wheat germ agglutinin-functionalized nanoparticles (WGA-NP) with various polymer architectures in the Caco-2 cell model. GFP-Rabs, Rab4, Rab5, Rab7, Rab11, GTPases served as key regulators of vesicular transport, and their mutants were transfected to Caco-2 cells respectively to determine the cellular itinerary of WGA-NP and the role of Rabs therein. Transcytosis inhibition experiments indicated that transcellular transport of WGA-NP (PEG(3000)-PLA(40000) formulation) happened in a cytoskeleton-dependent manner and majorly by means of clathrin-mediated mechanism. Intracellular transport, especially the endolysosome pathway was found largely contribute to the transcytosis of WGA-NP. WGA-NP with shorter surface PEG length (2000) resulted in higher cellular association and more colocalization with the clathrin-mediated transport pathway, while that with longer surface PEG length (5000) avoided the clathrin-mediated transport pathway but achieved higher transcytosis after 4?h incubation. WGA-NP with PLGA as the core materials obtained elevated lysosome escape and enhanced transcytosis after 2?h incubation. These findings provided important evidence for the role of polymer architectures in modulating cellular transport of functionalized nanocarriers, and would be helpful in improving carrier design to enhance drug delivery.