组织工程中生物可降解高分子的功能化及生物学修饰

可生物降解高分子在组织工程中有十分重要的作用。本文从组织工程对支架材料的生物学要求出发,对可生物吸收高分子的本体改性引入功能基团,进而通过化学键合促进细胞特异性黏附的短肽序列(ROD)、含功能性基团的水凝胶通过化学键合或物理包覆固定RGD以及聚合物表面修饰固定RGD三个方面进行了综述。     

具有双重抗菌作用的两性离子水凝胶

合成了一种甲基丙烯酰胺类两性离子单体,采用自由基聚合法制备了具有双重抗菌作用的水凝胶。利用1 H-NMR和FT-IR表征单体和水凝胶,考察了水凝胶释放水杨酸的动力学曲线。以导入绿色荧光蛋白的大肠杆菌为试验菌,荧光显微镜观察水凝胶表面细菌黏附情况;以金黄色葡萄球菌和大肠杆菌为试验菌,研究水凝胶抗菌性能。结果表明,该水凝胶可有效防止细菌黏附并且通过释放水杨酸实现抗菌功能。     

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.     

贻贝黏附蛋白启发的甲壳素纳米晶须增强湿态黏附水凝胶的研究

报道了一种力学性能优良,湿态生物组织黏附能高的黏附水凝胶.该凝胶由丙烯酸、甲基丙烯酸羟乙酯和3-三烯十五烷基-1,2-邻苯二酚共聚,与壳聚糖复合、并由甲壳素纳米晶须增强而成.该凝胶网络含有可逆和不可逆交联作用.其中可逆物理作用包括阴阳离子聚电解质静电吸引、烷基链疏水缔合、苯环π-π堆积、阳离子-π、氢键和拓扑纠缠.由这些物理键形成的次级网络的可逆形成/破坏为水凝胶形变提供了能量耗散,从而提升了其断裂韧性.另一方面,水凝胶的快速吸水能力破坏了湿润基体表面的水合层,使凝胶表面基团能与组织表面形成物理键和化学键的界面相互作用,从而共同促进水凝胶与湿态组织的强韧黏附.水凝胶的断裂强度可达276.4 kPa,对湿润猪皮的界面黏附韧性可达831 J/m²,在水下对猪皮的界面黏附韧性约达236 J/m²,猪皮和猪肝伤口闭合强度分别可达26.2和16.5 kPa.该黏附凝胶适合作为免缝合的伤口闭合黏胶材料.     

黄原胶纳米微凝胶的制备及其pH/还原响应性能

以胱胺四酰肼为交联剂,将其与黄原胶在水溶液中进行酰胺化反应,通过"一步法"制备得到pH和还原刺激响应性纳米微凝胶;采用傅里叶红外光谱仪、核磁共振氢谱仪、动态激光光散射仪、扫描电镜和透射电镜对其结构和形貌进行了表征,研究了纳米微凝胶的性能及其药物控释效果。结果表明:该纳米微凝胶具有明显的pH和还原响应性。纳米微凝胶中含有游离的酰肼基团,可与阿霉素分子中的酮羰基反应形成pH敏感的酰腙键。胱胺四酰肼中的双硫键可在较高浓度的谷胱甘肽作用下还原,导致微凝胶交联结构被破坏,促使药物释放。该纳米微凝胶生物相容性良好,有望用作靶向释放抗癌药物载体。     

具有双重抗菌作用的两性离子水凝胶

合成了一种甲基丙烯酰胺类两性离子单体,采用自由基聚合法制备具有双重抗菌作用的水凝胶。利用1 H-NMR和FT-IR表征单体和水凝胶,考察了水凝胶释放水杨酸的水解曲线。以导入绿色荧光蛋白的大肠杆菌为试验菌,荧光显微镜下观察水凝胶表面的细菌黏附情况;以金黄色葡萄球菌和大肠杆菌为试验菌,研究水凝胶的抗菌性能。结果表明,该水凝胶可有效防止细菌黏附并且通过释放水杨酸实现抗菌性能。     

表面接枝嵌段共聚物刷实现内皮细胞的选择性黏附

利用表面引发原子转移自由基聚合(SI-ATRP)技术将聚(甲基丙烯酸寡聚乙二醇酯)和聚(甲基丙烯酸缩水甘油酯)的双嵌段共聚物刷(POEGMA-b-PGMA)接枝在材料表面,并通过PGMA中丰富的环氧基团开环固定可特异性黏附内皮细胞的多肽GREDVY.静态水接触角、接枝层厚度、X射线光电子能谱(XPS)以及原子力显微镜(AFM)的测试结果证明了各步接枝反应的成功性.细胞培养研究表明这种连接有GREDVY的双嵌段共聚物表面能够有效的促进血管内皮细胞的黏附,同时排斥成纤维细胞的黏附,从而实现了内皮细胞的选择性黏附.     

超细荧光聚合物纳米微球的制备

合成了具有较高荧光量子产率(0.69)和良好光稳定性的可聚合荧光染料单体,该荧光染料的光稳定性高于商品化的染料罗丹明B。 通过氧化还原引发剂引发乳液聚合制备了超细荧光聚合物纳米微球,将染料分子共价连接在聚合物链上。 使用非离子表面活性剂Triton X-100作为乳化剂、甲基丙烯酸甲酯(MMA)作为单体和助乳化剂,制备的超细纳米微球平均粒径为22 nm,而不加MMA时制备出的纳米微球平均粒径在150 nm左右。 由于微球表面带有苄氯基团,为进一步的微球功能化提供了途径。     

尺寸可控的固定化漆酶-介体凝胶小球微反应器的制备和性能评价

首次利用漆酶-乙酰丙酮(AA)-过硫酸钾组成的复合引发体系在室温水相中引发丙烯酰胺的聚合, 采用滴球法将上述反应液滴入硫酸铜溶液, 利用铜离子与壳聚糖的络合交联反应制备得到尺寸可控的核壳结构小球. 小球内部发生自由基聚合反应, 将漆酶-AA同步固定于新生成的具有三维网络结构的水凝胶中. 微反应器小球的形貌和内部孔道结构通过扫描电镜和氮气吸附实验进行了表征. 该固定化漆酶-介体小球微反应器相比于游离漆酶表现出更高的pH稳定性和耐热性. 得益于固定化AA的介导作用, 漆酶的可重复使用性能得到显著提升, 降解孔雀石绿的有效循环批次较游离漆酶-介体体系延长了3倍, 为降低漆酶在实际废水处理中的成本、削减游离介体带来的二次污染提供了一条有效途径.     

含可脱除侧基的功能化微凝胶及其在药物释放中的应用

环境敏感微凝胶由于其对外界刺激的快速响应能力在药物传输和释放领域得到广泛的关注.本文报道了一种侧链含可脱除基团的温敏微凝胶,并探讨了其在药物释放中的潜在应用.通过分子设计,合成出含侧链N-叔丁氧羰基(N-Boc)的疏水单体N-(N-叔丁氧羰基-乙二胺)甘氨酸二肽甲基丙烯酰胺(BEMAGG),然后将其与N-异丙基丙烯酰胺(NIPAAm)沉淀共聚合直接得到侧链含Boc基团的微凝胶MG-Boc.通过浊点法、粒径和Zeta电位测定研究了微凝胶中Boc基团在酸中的脱除过程及其对微凝胶性能的影响,研究表明Boc基团的脱除显著影响微凝胶的体积相转变温度、粒径和Zeta电位.对盐酸阿霉素药物的释放研究表明,释放明显依赖于释放介质的pH值.该响应性的微凝胶在药物控制释放领域具有潜在的应用前景.     

Circulating IgSF Proteins Inhibit Adhesion of Antibody Targeted Microspheres to Endothelial Inflammatory Ligands

Proposed methods for detecting circulatory system disease include targeting ultrasound contrast agents to inflammatory markers on vascular endothelial cells. For antibody-based therapies, soluble forms of the targeted adhesion proteins of the immunoglobulin superfamily (IgSF) reduce adhesion yet were left unaccounted in prior reports. Microspheres labeled simply with a maximum level of antibodies can reduce the diagnostic sensitivity by adhering to proteins expressed normally at a low level, while sparsely coated particles may be rendered ineffective by circulating soluble forms of the targeted proteins. A new microdevice technique is applied to simultaneously measure the adhesion profile to a series of IgSF-protein-coated surfaces. In this investigation, we quantify the in vitro binding characteristics of 5-μm microspheres to oriented intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein-coated surfaces in the presence of human serum at physiological concentrations. Defined regions of a slide were coated with recombinant chimeric Fc-human ICAM-1 and VCAM-1 in variable ratios but constant total concentration. Monoclonal human anti-ICAM-1 or anti-VCAM-1 antibodies in competition with non-binding mouse anti-rabbit antibodies coat the microsphere surface at a constant surface density with variable yet controlled surface activities. Using multiple slide surface IgSF protein and microsphere antibody concentrations, an adhesion profile was developed for the microspheres with and without IgSF proteins from human serum, which demonstrated that exposure to serum reduced microsphere binding, on average, more than 50% compared to the no-serum condition.. The serum effects were limited to antibodies on the microsphere, since binding inhibition was reversed after rinsing serum from the system and fresh antibody-coated microspheres were introduced. This analysis quantifies the binding effects of soluble IgSF proteins from human serum on antibody-based targeted ultrasound detection and drug delivery methods.     

Covalent immobilization of p-selectin enhances cell rolling

Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.     

New biodegradable polymers for delivery of bioactive agents

Biodegradable, thermosensitive triblock copolymer, PLGA-PEG-PLGA, can be easily fabricated into drug-loaded microspheres or injectable in situ hydrogel system for protein or water-insoluble drugs without use of organic solvent. Aqueous-based microsphere exhibited continuous release of intact insulin in vitro for 3 weeks while the microspheres prepared using dichloromethane showed initial burst and incomplete release. Confocal miscoscopy images of microspheres corroborated the release pattern. Next study with an injectable in situ hydrogel (ReGel^TM) exhibited zero-order insulin release in vitro and sustained plasma insulin level for 2 weeks in vivo upon single subcutaneous injection in SD rats.     

Hydrogel-shelled biodegradable microspheres for sustained release of encapsulants

Biodegradable microparticles are promising for the sustained release of encapsulated lipophilic drugs. In particular, the microparticles with uniform size show excellent linearity of cumulated release over time with minimized initial burst. Here, we encapsulate the biodegradable microparticles with a hydrogel shell to improve the controllability over the sustained release and suspension stability. With a capillary microfluidic device, monodisperse oil-in-water-in-oil (O/W/O) double-emulsion droplets are produced to have a toluene solution of polylactic acid (PLA) in the core and sodium alginate and calcium-ethylenediaminetetraacetic acid (EDTA) complex in the shell, whereas the continuous oil phase contains acetic acid. As the toluene evaporates, PLA consolidates to form a microsphere in the core. At the same time, acetic acid diffuses from the continuous phase to the water layer, which causes the dissociation of the Ca-EDTA complex and the gelation of alginate. The hydrogel-shelled PLA microspheres are transferred from the oil to an aqueous solution of calcium chloride, which further tightens the gel shell. The resulting core-shell microspheres show sustained release of encapsulants for extended periods as the hydrogel shell serves as a diffusion barrier. Moreover, the hydrogel shells prevent interparticle agglomeration and adhesion to the solid walls, securing high suspension stability during the injection.     

Synthesis and Reactions of Hydrophilic Functional Microspheres for Immunological Studies

There is a need for hydrophilic polymeric microspheres with functional groups on their surface which can be reacted efficiently with proteins. These microspheres with antibodies (immunoglobulins) covalently bound to their surfaces constitute valuable immunoreagents capable of marking specific receptors (antigens) on cell surface membranes. The main requirements of the microspheres for the above applications are: insolubility in aqueous or organic media, absence of aggregation and of nonspecific interaction with cells and presence of suitable functional groups for covalent binding with antibodies. Hydrophobic polystyrene or poly(methyl methacrylate) latices do not meet these requirements. Copolymerization of hydrophilic monomers under suitable experimental conditions yielded microspheres with the required characteristics. Emulsion polymerization and ionizing radiation were found to constitute convenient techniques for the synthesis of hydrophilic and crosslinked (and therefore insoluble) functional microspheres ranging in diameter from 0.01 to 8 μm. By choosing suitable comonomers, it was possible to incorporate hydroxyl, carboxyl, amido, and dimethylamino functional groups into the particles. Copolymerization with isomeric vinylpyridines or dimethylamino methacrylate yielded weakly or strongly basic groups, respectively, capable of binding with acids. The experimental conditions suitable for obtaining desired particle sizes, in a relatively narrow distribution, were determined. It was found that the particle size depended to a large extent on the water solubility of the monomers, the presence or absence of stabilizer, the concentration of a surfactant, and the monomer concentration. The preferred technique to bind antibodies to the microsphere surface consisted of reacting amino groups with glutaraldehyde followed by the reaction with proteins. The use of polyglutaraldehyde instead of glutaraldehyde was also investigated. For this purpose the rate of polymerization of glutaraldehyde as a function of concentration and pH was first studied, followed by a study of the reactivity of polyglutaraldehyde micro-spheres with immunoglobulins. A recent new development of importance for cell separation is the synthesis of functional microspheres containing magnetic iron oxide. Preliminary investigations show that red blood cells and lymphocytes labeled with magnetic immunomicrospheres can be efficiently separated by means of permanent magnet. Separation of labeled from unlabeled human red blood cells was also achieved by means of a free-flow electrophoretic instrument.     

Interactions between temperature-sensitive hydrogel microspheres and granulocytes

Poly(N-isopropylacrylamide) (PNIPAM) has a low critical solution temperature (LCST) at 32°C in water and the hydrophilicity changes through the LCST. The microspheres whose surface was composed of PNIPAM exhibited phase transition behavior around 32°C. Therefore, the interactions between PNIPAM micropheres and granulocytes depended on the temperature. That is, the oxygen consumption and active oxygen production by cells in contact with PNIPAM-containing microspheres and adhesion of the microspheres to the cell surface were more enhanced above the LCST of PNIPAM than below it, whereas no significant temperature dependence of cell–microspheres interaction was observed in nonthermosensitive microsphere systems. It was suggested that the function of cells could be controlled with temperature using the temperature-sensitive microspheres.     

Preparation of Porous Polylactide Microspheres and Their Application in Tissue Engineering

In this study,porous polylactide (PLA) microspheres with different structures were prepared through the multiple emulsion solvent evaporation method.By changing organic solvents (ethyl acetate and chloroform) and adding effervescent salt NH4HCO3 in the inner water phase,microspheres with porous capsular,matrix,microcapsular and multivesicular structures were prepared.The protein encapsulation and release,and the cell growth behavior of porous microspheres were further explored.Under the same inner water phase,microspheres prepared with chloroform had higher protein encapsulation efficiency and less protein release rate as compared with those prepared with ethyl acetate.Cell experiments showed that the relatively rough surface of microspheres prepared with chloroform was more favorable for the cell growth in comparison with the smooth surface of microspheres prepared with ethyl acetate.This study shows a simple and effective method to control the protein release and cell growth behaviors of polymer microspheres by tuning their porous structure.     

基于天然高分子基元的阻隔层对磁性载药聚乳酸微球的控释作用

利用层层组装技术构建了基于天然高分子壳聚糖和海藻酸钠的阻隔层, 并研究了该阻隔层对磁性载药聚乳酸微球的药物释放作用. 实验结果表明, 阻隔层能够有效抑制模型药物的突释, 具有延缓药物释放的效果. 具有阻隔层的磁性载药体系具有药物释放平缓和生物相容性高等特点, 是理想的磁靶向载药体系.     

Thermoresponsive and bioactive poly(vinyl ether)-based hydrogels synthesized by radiation copolymerization and photochemical immobilization

A thermoresponsive hydrogel was synthesized by radiation copolymerization of ethylene glycol vinyl ether (EGVE) and butyl vinyl ether (BVE) in the presence of cross-linking agent diethylene glycol divinyl ether. The gel was modified by a cell adhesion factor RGD by photochemical immobilization technique. While the unmodified hydrogel shows fully hydrated form at low temperatures (+4 °C) and it extensively dehydrates at 37 °C, the biomodified hydrogel still kept its thermoresponsive character after immobilization. The effectiveness of immobilization was checked with FTIR-ATR and XPS. The use of bioactive thermoresponsive hydrogels in cell culture applications was investigated. For this purpose, cell culture experiments were realized by L929 mouse fibroblasts. Cell attachment experiments revealed the effect of immobilized RGD with higher values of cell attachment (∼85%), which were obtained especially in the absence of serum. The thermoresponsive character of the hydrogel was useful for the application of low-temperature treatment in order to recover the attached viable cells from the surface of the hydrogel without using trypsin. When the culture temperature was decreased from 37 to 10 °C for 30 min ∼80% of the cells were detached from the hydrogel surface.     

Preparation and Characterization of Magnetic Targeted Drug Controlled-Release Hydrogel Microspheres

Magnetic targeted drug controlled release hydrogel microspheres were prepared by a radiation technique. Ferric oxide granules (size around 50 nm) were used as the core for magnetic target. The PVP ferrogels (ferromagnetic nanoparticles in hydrogel microsphere) were obtained by irradiating an emulsion of Poly (N-vinylpyrrolidone) PVP/ferromagnetic granule with cobalt 60 γ–ray. The morphology of the PVP ferrogel was studied by both optical and electronic microscopy, respectively. A broad-spectrum anticancer drug, Bleomycin A5 Hydrochloride (BLM), was immobilized in the ferrogel and the release property of the drug in vitro was studied. The function of targeting and anti-cancer was studied on the New Zealand White rabbits, based on the implantation of experimental VX2 squamous cell carcinoma in the auricles of the rabbits.