功能化聚乳酸微球改性聚乳酸膜片表面及其细胞相容性

以两亲三嵌段共聚物PEO PPO PEO及其氨基酸、多肽RGD衍生物作为微球制备过程中的表面稳定剂 ,低分子量的聚乳酸作为成核材料 ,采用o w乳液溶剂挥发法制备得到表面含不同氨基酸的聚乳酸微球 ,荧光标记手段证明了氨基酸存在于微球的表面 ;利用溶胀嵌入固定法将微球固定到具有生物惰性聚乳酸膜片表面 ,制备得到了具有良好稳定性的微球改性聚乳酸生物惰性膜片 ;成骨细胞相容性测试表明 ,氨基酸尤其是多肽RGD在生物惰性聚乳酸表面的引入能较大程度地提高聚乳酸生物惰性表面的细胞相容性 ,能较好地诱导细胞行为如细胞粘附、增殖等过程的发生 ,为实现对细胞行为的诱导控制提供了一种新途径 .     

一种仿细胞膜聚合物用于碳纳米管表面改性

以新型含有磷酸胆碱基的仿细胞膜两亲聚合物——胆固醇封端的聚(2-甲基丙烯酰氧基乙基磷酸胆碱)(CPMPC)为表面稳定剂实现碳纳米管的表面改性,利用两亲聚合物中的胆固醇疏水段与碳纳米管表面进行非共价键的稳定结合,通过两亲聚合物中聚(2-甲基丙烯酰氧基乙基磷酰胆碱)(PMPC)亲水段实现其水溶性和生物相容性.并以商业可获得的典型两亲分子,末端为胆固醇的聚氧乙烯(CPEG)和卵磷脂,为对照进行研究.研究表明CPMPC和CPEG均具有比卵磷脂更高的对碳纳米管进行分散的能力.而CPMPC改性的碳纳米管比CPEG改性的碳纳米管具有更优的稳定性和生物相容性,通过新型仿细胞膜聚合物改性的碳纳米管在生物医用领域有潜在应用.     

基于聚乙二醇和不同多肽构建的生物涂层及其内皮细胞选择性研究

利用自由基聚合反应将甲基丙烯酸聚乙二醇酯(PEGMA)和甲基丙烯酸缩水甘油酯(GMA)的二元共聚物接枝在基材表面,并通过开环反应分别固定精氨酸-甘氨酸-天冬氨酸(RGD)、精氨酸-谷氨酸-天冬氨酸-缬氨酸(REDV)和酪氨酸-异亮氨酸-甘氨酸-丝氨酸-精氨酸(YIGSR)3种可特异性黏附内皮细胞的多肽.通过核磁共振检测合成的聚合物分子结构,并进一步通过X射线光电子能谱(XPS))以及原子力显微镜(AFM)的测试结果证明聚合物成功接枝在基材表面.利用紫外-可见吸收光谱(UV-Vis)对表面固定的3种多肽进行了定量表征.体外内皮细胞和平滑肌细胞黏附结果表明,3种不同多肽修饰的共聚物表面均能够有效阻抗平滑肌细胞的黏附,同时不同程度地促进内皮细胞的黏附,从而实现了基材表面内皮细胞的选择性黏附.其中与RGD和YIGSR多肽修饰的表面相比,REDV多肽修饰的表面呈现出更优异的内皮细胞选择性.这种具有内皮细胞特异选择性的界面在心血管支架涂层原位内皮化方面具有良好的应用前景.     

利用表面上的小分子控制细胞黏附

细胞黏附是重要的生理过程,多细胞生物体中大部分种类的细胞都依赖于在表面的黏附而进行其正常生理活动。细胞的黏附需要固定在表面的有机分子（例如蛋白质或多肽）作配体。我们利用表面小分子模拟蛋白质或多肽作为配体,通过与细胞膜上受体结合,促进细胞黏附到表面。聚乙二醇（PEG）可以抵抗细胞在表面的黏附,我们利用含有PEG的表面小分子来调节细胞黏附。细胞表面的受体与胞外基质表面的配体结合是一个动态过程,在适宜时间和空间发生的时候,细胞就会产生运动和迁移,细胞的迁移也是重要的生理过程。本文主要介绍近年来利用小分子的表面化学和微纳米结构控制细胞在表面的黏附和迁移。     

聚甲基丙烯酸甲酯/星形聚乙二醇半互穿聚合物网络的形状记忆性能和分子机理

采用非共价复合方法,设计并合成了具有星形结构的聚甲基丙烯酸甲酯/星形聚乙二醇半互穿聚合物网络(PMMA/SPEG)和聚甲基丙烯酸甲酯/线性聚乙二醇半互穿聚合物网络(PMMA/LPEG).研究了PEG分子量对PMMA/SPEG和PMMA/LPEG的热性能、机械性能、动态机械性能和形状记忆性能的影响.结果表明,与PMMA/LPEG相比,星形结构的嵌入显著提高了PMMA/SPEG复合物的机械性能、形状回复率和回复速度.采用Edwards管道模型理论对其形状记忆效应的分子机理进行了阐释,利用材料的应力松弛特性对机理分析进行了验证.     

PEG链段对聚乙二醇接枝聚苯胺结构与性能的影响

将苯胺(An)与甲氧基聚乙二醇邻氨基苯基醚氧化共聚,制备了梳状接枝共聚物PAn-g-PEG.研究了梳状接枝共聚物的UV-Vis、微观结构、热稳定性和溶解成膜性等随侧链聚乙二醇(PEG)链段的变化规律.结果表明随PAn-g-PEG中PEG链段长度和含量的提高,共聚物的溶解性和成膜性能显著提高,电子导电率缓慢降低,热稳定性变差.共聚物具有微相分离结构,其形态随PEG链段的改变分别为“海-岛相”和“双连续相”;提高PEG链段长度和含量,PAn-g-PEG能形成稳定的水溶性分散体系,并能浇注成柔韧平整的导电高分子自支撑膜.     

基于聚乙二醇结构调控高分子量聚乳酸立构复合体系的结晶行为

设计合成了梳形聚(聚乙二醇甲醚丙烯酸酯)(PPEGA)及其与聚乙二醇(PEG)的嵌段共聚物(PEG-b-PPEGA).通过与高分子量左旋聚乳酸(PLLA)和右旋聚乳酸(PDLA)共混探究了PEG不同的结构对PLA立构复合体系(sc-PLA)结晶的影响.结果 表明线形PEGA和PEG能与sc-PLA完全相容,两者均能促进...     

PLG-g-TA/RGD酶催化交联水凝胶用于透明软骨细胞黏附和三维细胞培养

制备了一种基于聚谷氨酸-g-酪胺/cRGDfk(PLG-g-TA/RGD)的新型酶催化交联水凝胶, 用于兔透明软骨细胞黏附和三维细胞的培养. PLG-g-TA/RGD聚合物材料在辣根过氧化物酶(HRP)和过氧化氢(H₂O₂)存在下, 能够通过酪氨基团的自交联快速形成水凝胶. 环状多肽(cRGDfk)的引入能够显著提高材料的溶液-凝胶转变速率和凝胶强度. 透明软骨细胞在水凝胶表面黏附3 d后, 在PLG-g-TA/RGD水凝胶表面有更多的细胞黏附; 将透明软骨细胞包裹在水凝胶内培养1, 4, 7 d后, 细胞在PLG-g-TA/RGD水凝胶内增殖效率明显高于对照组PLG-g-TA水凝胶. 细胞实验结果表明, 该水凝胶材料具有良好的生物相容性. cRGDfk的引入, 促进了透明软骨细胞的黏附和增殖, 显示了PLG-g-TA/RGD水凝胶材料在三维细胞培养方面的应用潜力.     

高密度自由醛基聚合物粉体材料的制备与应用

在酸性条件和剧烈搅拌下, 将质量分数为2.0%的聚乙烯醇水溶液缓慢滴加到过量25%(质量分数)的戊二醛水溶液中, 得到的产物不是凝胶, 而是粒度在50～150 nm之间的刚性粉末. 该粉末体相和表面依然保留着高密度的自由醛基. 理论计算得出粉末中整体醛基的量大于5.5 mmol/g, 而实验测得表面醛基的量大于1.6 mmol/g. 将醛基粉体材料进一步和氨基化合物反应, 其官能团分别转化为—NH2, —SH和—NH—NH2. 这类新型的大分子化合物普遍具有三维的亲水性表面、足够数量且灵活的功能性官能团和低的荧光背景, 是制备3D生物芯片的优良材料. 其中, 醛基和酰肼基粉末对肝素具有良好的固定效果, 固定的肝素分子依然具有较高的抗凝血活性.     

聚氯乙烯表面共价键合肝素及抗凝血性的研究

采用Ar等离子体引发聚乙二醇(PEG)在聚氯乙烯(PVC)表面固定化,进一步对固定PEG后的PVC进行肝素化处理,以改善PVC材料的抗凝血性能。探讨了PEG浓度对Ar等离子体固定化反应效果的影响。通过X射线光电子能谱(XPS)、衰减全反射红外光谱(ATR-FTIR)、扫描电镜(SEM)和接触角测定研究了固定PEG前后PVC的表面性能和表面形貌的变化。XPS分析证实肝素已成功地共价键合于PVC表面。采用体外凝血时间测定和血小板粘附实验对材料的抗凝血性能进行评价,结果表明,被修饰PVC材料的抗凝血性能显著提高。     

Different complex surfaces of polyethyleneglycol (PEG) and REDV ligand to enhance the endothelial cells selectivity over smooth muscle cells

Arg-Glu-Asp-Val (REDV) peptide with endothelial cells (ECs) selectivity was immobilized onto PEG based polymeric coating via the active p-nitrophenyloxycarbonyl group. The adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) onto surface modified either by REDV end-tethered polyethylene glycol (PEG) or by the complex of free PEG and REDV were investigated to understand the synergic action of nonspecific resistance of PEG and specific recognitions of REDV. Cell culture results indicated that the surfaces end tethered by REDV peptide via PEG "spacer" (n=1, 6, 10) exhibited slight EC selectivity and showed small difference between different lengths of PEG chain. Both separate-culture and co-culture of HUVECs and HASMCs indicated that the introducing of free PEG into REDV tethered surface inhibited HASMCs adhesion significantly and remained a high level of HUVECs growth. Furthermore, the surface with short free PEG chain (n=6) was much more effective to enhance ECs selectivity than long EG chain (n=23). The combination of nonspecific resistance of short free PEG and the ECs selectivity of REDV peptide presents much better ability to enhance the competitive adhesion of HUVECs over HASMCs.     

Surface-grafted polystyrene beads with comb-like poly(ethylene glycol) chains: preparation and biological application

We prepared surface-grafted polystyrene (PS) beads with comb-like poly(ethylene glycol) (PEG) chains. To accomplish this, conventional gel-type PS beads (35-75 microm) were treated with ozone gas to introduce hydroperoxide groups onto the surface. Using these hydroperoxide groups, poly(methyl methacrylate) (PMMA, Mn= 22,000-25,000) was grafted onto the surface of the PS beads. The ester groups of the grafted PMMA were reduced to hydroxyl groups with lithium aluminum hydride (LAH). After adding ethylene oxide (EO) to the hydroxyl groups, we obtained the PS-sg-PEG beads, which had a rugged surface and a diameter of 80-150 microm. We could obtain several kinds of the PS-sg-PEG beads by controlling the chain lengths of the grafted PMMA and the molecular weights of the PEG chains. The grafted PEG layer was about 30-50 microm thick, which was verified from the cross-sectioned views of the fluorescamine-labeled beads. These fluorescence images proved that the beads possessed a pellicular structure. Furthermore, we found that the surface-grafted PEG chains had the characteristic property of reducing non-specific protein adsorption on the beads.     

Characterization of aldehyde-PEG tethered surfaces: influence of PEG chain length on the specific biorecognition

A functionalized poly(ethylene glycol) (PEG) layer possessing a reactive aldehyde group at the free end of the tethered PEG chain was constructed by simple coating on the substrate, using the acetal-PEG/poly(DL-lactide) block copolymer, followed by the hydrolysis of the acetal end group by an acid treatment. The reactivity of the aldehyde group at the distal end of the PEG tethered chain was evaluated via a reductive amination using 4-amino-2,2,6,6-tetramethylpiperidinyloxy as the model compound. Further conjugation of the aldehyde group with sugar moieties has demonstrated an increased recognition ability with lectins with an increasing PEG chain length, which was attributable to the mobility of the chain end. These results provide a novel idea for highly sensitive biorecognition, suggesting a method to create highly selective biosensing surfaces that are able to prevent the undesired nonspecific adsorption of biocomponents.     

RGD改性聚醚氨酯及其内皮细胞相容性的研究

利用氢键稳定的溶液互穿技术对聚醚氨酯(PEU)进行改性.用ATR-FTIR对十八烷基-聚氧乙烯-4,4'-二苯甲烷二异氰酸酯-聚氧乙烯-十八烷基(MSPEO)与PEU共混膜表面进行研究,结果表明,MS-PEO中的氨基甲酸酯链段与PEU基材之间发生了氢键缔合的作用.通过水化处理PEO及十八烷基自发地富集在基材表面.根据氢键缔合和表面自迁移原理,设计了两种RGD改性聚醚氨酯的方法:(1)将含RGD端基的聚氧乙烯-4,4'-二苯甲烷二异氰酸酯-聚氧乙烯偶联物(MPEO-RGD)与PEU进行共混改性,利用RGD端基及PEO的自迁移特性获得RGD富集的表面;(2)将含甲磺酸酯端基的聚氧乙烯-4,4'-二苯甲烷二异氰酸酯-聚氧乙烯偶联物(MPEO-mesyl)与PEU共混成膜,并对膜片进行水化处理,使甲磺酸酯端基富集在PEU表面,浸泡于RGD的PBS溶液中,在膜片表面成功地原位接枝了RGD.对两种RGD改性方法获得的表面进行了内皮细胞的培养,结果表明,两种改性方法均大大提高了PEU的细胞相容性,其中方法(1)共混改性的表面细胞相容性略优于方法(2)的接枝改性表面.     

Structure and thermodynamics of lipid bilayers on polyethylene glycol cushions: fact and fiction of PEG cushioned membranes

In developing well hydrated polymer cushioned membranes, structural studies are often neglected. In this work, neutron and X-ray reflectivity studies reveal that hybrid bilayer/polyethylene glycol (PEG) systems created from mixtures of phospholipids and PEG conjugated lipopolymers do not yield a hydrated cushion beneath the bilayer unless the terminal ends of the lipopolymers are functionalized with reactive end groups and can covalently bind (tether) to the underlying support surface. While reactive PEG tethered systems yielded bilayers with near complete surface coverage, a bimodal distribution of heights with sub-micrometer lateral dimensions was observed consisting of cushioned membrane domains and uncushioned regions in close proximity to the support. The membrane fraction cushioned by the hydrated polymer could be controlled by adjusting the molar ratio of lipopolymer in the bilayer. A general phase diagram based on the free energy of the various configurations is derived that qualitatively predicts the observed behavior and the resulting structure of such systems a priori. As further evidenced by ellipsometry, atomic force and fluorescence microscopy, the tethered system provides a simple means for fabricating small cushioned domains within a membrane.     

Pattern generation of biological ligands on a biodegradable poly(glycolic acid) film

The micropatterns of biological ligands (biotin and RGD peptides) were generated on a flat surface of biodegradable polymer, poly(glycolic acid) (PGA). The immobilization of biological ligands onto the surface of biodegradable polymers (especially aliphatic polyesters) is usually hampered by the absence of functionalizable groups on the polymer backbone. We demonstrate herein that PGA polymer films were modified by surface hydrolysis to introduce carboxylic acid groups on the film surfaces, which were subsequently used for patterning amine-terminated ligands by microcontact printing. Fluorescence microscopy was used to verify the pattern of biotin on the surface of the PGA films after complexation with fluorescein-conjugated streptavidin. In addition, the cellular micropatterns were obtained from micropatterns of RGD peptides on the surface-hydrolyzed PGA films.     

Buried, covalently attached RGD peptide motifs in poly(methacrylic acid) brush layers: the effect of brush structure on cell adhesion

Iniferter-mediated surface-initiated photopolymerization was used to graft poly(methacrylic acid) (PMAA) brush layers obtained from surface-attached iniferters in self-assembled monolayers to a gold surface. The tethered chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) motif. The modified brushes were extended by reinitiating the polymerization to obtain an additional layer of PMAA, thereby burying the peptide-functionalized segments inside the brush structure. Contact angle measurements and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the wettability and the chemical properties of these platforms. Time of flight secondary ion mass spectroscopy (TOF-SIMS) measurements were performed to monitor the chemical composition of the polymer layer as a function of the distance to the gold surface and obtain information concerning the depth of the RGD motifs inside the brush structure. The brush thickness was evaluated as a function of the polymerization (i.e., UV-irradiation) time with atomic force microscopy (AFM) and ellipsometry. Cell adhesion tests employing human osteoblasts were performed on substrates with the RGD peptides exposed at the surface as well as covered by a PMAA top brush layer. Immunofluorescence studies demonstrated a variation of the cell morphology as a function of the position of the peptide units along the grafted chains.     

DNA‐Catalyzed Introduction of Azide at Tyrosine for Peptide Modification

We show that DNA enzymes (deoxyribozymes) can introduce azide functional groups at tyrosine residues in peptide substrates. Using in vitro selection, we identified deoxyribozymes that transfer the 2′‐azido‐2′‐deoxyadenosine 5′‐monophosphoryl group (2′‐Az‐dAMP) from the analogous 5′‐triphosphate (2′‐Az‐dATP) onto the tyrosine hydroxyl group of a peptide, which is either tethered to a DNA anchor or free. Some of the new deoxyribozymes are general with regard to the amino acid residues surrounding the tyrosine, while other DNA enzymes are sequence‐selective. We use one of the new deoxyribozymes to modify free peptide substrates by attaching PEG moieties and fluorescent labels.     

Demonstration of α-helical structure of peptides tethered to gold surfaces using surface infrared and circular dichroic spectroscopies

Gold and quartz surfaces terminated in an alkane thiol self-assembled monolayer (SAM) that were partially terminated with azide were reacted with a helical peptide containing two alkyne groups in a Cu(I)-catalyzed Huisgen cycloaddition. Surface grazing incidence angle reflection-absorption infrared spectroscopy (GRAS-IR) was used to determine that when the Au surface was terminated with 25% of the monolayer containing azide groups, 92% of available azide groups reacted with the peptide. The majority of peptides reacted with both alkynes, resulting in peptides tethered to the surface through two covalent bonds. This was confirmed by comparison to a control peptide containing only one reactive alkyne group. Surface circular dichroic (CD) spectroscopy showed that while the helical structure of the peptide was distorted in the reaction solution, α-helical structure was induced when tethered on the SAM functionalized Au surface. Demonstration of the preservation of desired secondary structure of helical elements at a chemically functionalized surface is an important advance in preparing robust biologically mimetic surfaces to integrate functioning proteins into inorganic materials.     

Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells

Osteogenic differentiation and mineralization of bone marrow stromal (BMS) cells depends on the cells' interactions with bioactive peptides associated with the matrix proteins. The RGD peptides of ECM proteins interact with BMS cells through integrin surface receptors to facilitate cell spreading and adhesion. The BMP peptide corresponding to residues 73-92 of bone morphogenetic protein-2 promotes differentiation and mineralization of BMS cells. The objective of this work was to investigate the effects of RGD and BMP peptides, grafted to a hydrogel substrate, on osteogenic differentiation and mineralization of BMS cells. RGD peptide was acrylamide-terminated by reacting acrylic acid with the N-terminal amine group of the peptide to produce the functionalized Ac-GRGD peptide. The PEGylated BMP peptide was reacted with 4-carboxybenzenesulfonazide to produce an azide functionalized Az-mPEG-BMP peptide. Poly (lactide-co-ethylene oxide- co-fumarate) (PLEOF) macromer was cross-linked with Ac-GRGD peptide and propargyl acrylate to produce an RGD conjugated hydrogel. Az-mPEG-BMP peptide was grafted to the hydrogel by "click chemistry". The RGD and BMP peptide density on the hydrogel surface was 1.62+/-0.37 and 5.2+/-0.6 pmol/cm2, respectively. BMS cells were seeded on the hydrogels and the effect of RGD and BMP peptides on osteogenesis was evaluated by measuring ALPase activity and calcium content with incubation time. BMS cells cultured on RGD conjugated, BMP peptide grafted, and RGD+BMP peptide modified hydrogels showed 3, 2.5, and 5-fold increase in ALPase activity after 14 days incubation. BMS cells seeded on RGD+BMP peptides modified hydrogel showed 4.9- and 11.8-fold increase in calcium content after 14 and 21 days, respectively, which was significantly higher than RGD conjugated or BMP grafted hydrogels. These results demonstrate that RGD and BMP peptides, grafted to a hydrogel substrate, act synergistically to enhance osteogenic differentiation and mineralization of BMS cells. These findings are potentially useful in developing engineered scaffolds for bone regeneration.