Designed synthesis of fluorous‐functionalized magnetic mesoporous microspheres for specific enrichment of phosphopeptides with fluorous derivatization

In this work, for the first time, perfluorinated magnetic mesoporous microspheres were designed and synthesized for the highly specific enrichment of fluorous‐derivatized phosphopeptides through the unique fluorine–fluorine interactions. The perfluorinated magnetic mesoporous microspheres were prepared through a surfactant‐mediated one‐pot approach and successfully applied to the selective extraction of fluorous‐derivatized phosphopeptides from β‐casein tryptic digest, protein mixtures, and human serum. Thanks to the hydrophilic silanol groups exposed on the surface, perfluorinated groups modified in the pore channels and the magnetic cores, the flourous‐functionalized magnetic microspheres exhibited excellent dispersibility, specificity toward fluorous‐derivatized phosphopeptides while facilitated separation procedures. The novel composites achieved a high selectivity of 1:1000 toward nonphosphorylated peptides and proved to be practicable in the enrichment of endogenous phosphopeptides in the human serum sample.     

担载b-FGF的PLGA微球复合明胶支架的体外释放研究

目的:研究担载碱性成纤维细胞生长因子(b-FGF)微球复合明胶支架的外形特征、孔径、孔隙率及体外释放动力学,以期构建具有缓释功能、高孔隙率的担载细胞因子的新型复合明胶支架。方法:本文利用冷冻相分离法和S/O/W法先将b-FGF水溶液包裹于PLGA微球中,然后埋置于明胶溶液中制备为多孔复合明胶支架。分别对微球的形态和复合明胶支架的基本形态、孔径、孔隙率进行表征,通过Elisa法测定b-FGF在复合明胶支架中的体外释放行为。结果:制备成形态良好的三维复合明胶支架,其孔隙率为82.90%±1.45%,孔径范围为150~300μm,复合明胶支架中b-FGF在体外缓慢释放20余天。结论:担载蛋白微球复合明胶支架不仅满足组织工程支架的要求,还能有效缓释细胞因子,为细胞和组织生长提供良好的微环境,为进一步应用于组织工程领域提供了可能。     

Synthetic hydrogel microspheres as substrata for cell adhesion and growth

Summary Cross-linked poly(methyl methacrylate) (PMMA) microspheres were subjected to alkaline hydrolysis to obtain hydrophilic microspheres having carboxyl residues distributed throughout the matrix. These microspheres were found to support the growth of human skin fibroblasts and human heart and lung cells. Further, fibroblasts grown on them were found to be comparable with those grown on the commercial tissue culture plate with respect to [¹⁴C]amino acid uptake and incorporation into proteins. The hydrolyzed PMMA microspheres may find application as a microcarrier for cell culture. This work was supported in part by the Indian Council of Medical Research, New Delhi.     

Morphological aspects of interactions between microparticles and mammalian cells: intestinal uptake and onward movement

Uptake of ingested microparticles into small intestinal tissues and on to secondary organs has moved from being an anecdotal phenomenon to a recognised and quantifiable process, which is relevant to risk assessment of accidental exposure, treatment of multi-organ dysfunction syndrome and therapeutic uses of encapsulated drug or vaccine delivery. This review puts in context with the literature the findings of a morphological study of microparticle uptake, using two approaches.The first is a rat in vivo in situ model, appropriate to a study rooted in the exposure of human populations to microparticles. Latex microspheres 2 μm in diameter are the principal particle type used, although others are also investigated. Most data are based on microscopy, but analysis of macerated bulk tissue is also useful. Uptake occurs at early time points after a single dose and is shown to take place almost entirely at villous rather than Peyer's patch sites: however, multiple feeding and therefore a longer time-span produces a higher proportion of particles associated with Peyer's patches, albeit for very small total uptake at those later time points. Uptake is less affected by species, fasting and immunological competence than by age and reproductive status.The second approach uses in vitro methods to confirm the role of intercellular junctions in particle uptake. Particle-associated tight junction opening, in a Caco-2 monolayer, is reflected in changes in transepithelial resistance and particle uptake across the epithelial monolayer: Tight junction opening and particle uptake are both increased further by external irradiation, ethanol and sub-epithelial macrophages, but reduced by exposure to ice. An M cell model has looser tight junctions than Caco-2 cells, but a similar level of particle uptake. These results, along with the changes seen in junctional proteins after particle addition, confirm the role of tight junctions in uptake but suggest that adhering junctions are also important.     

Preparation of magnetic core-mesoporous shell microspheres with C8-modified interior pore-walls and their application in selective enrichment and analysis of mouse brain peptidome

In this paper, magnetic mesoporous silica microspheres with C8-modified interior pore-walls were prepared through a facile one-pot sol-gel coating strategy, and were successfully applied for selective enrichment of endogenous peptides in mouse brain for peptidome analysis. Through the one-pot sol-gel approach with surfactant (CTAB) as a template, tetraethyl orthosilicate (TEOS) and n-ctyltriethoxysilane (C8TEOS) as the precursors, C8-modified magnetic mesoporous microspheres (C8-Fe(3)O(4)@mSiO(2)) consisting magnetic core and mesoporous silica shell with C8-groups exposed in the mesopore channels were synthesized. The obtained microspheres possess highly open mesopores of 3.4 nm, high surface area (162.5 m(2)/g), large pore volume (0.17 cm(3)/g), excellent magnetic responsivity (56.3 emu/g) and good dispersibility in aqueous solution. Based on the abundant surface silanol groups, functional C8 groups and the strong magnetic responsivity of the core-shell C8-Fe(3) O(4) @mSiO(2) microspheres, efficient and fast enrichment of peptides was achieved. Additionally, the C8-Fe(3)O(4)@mSiO(2) microspheres exhibit excellent performance in selective enrichment of endogenous peptides from complex samples that are consist of peptides, large proteins and other compounds, including human serum and mouse brain followed by automated nano-LC-ESI-MS/MS analysis. These results indicate C8-Fe(3)O(4)@mSiO(2) microspheres would be a potential candidate for endogenous peptides enrichment and biomarkers discovery in peptidome analysis.     

装载于PLGA中的5-氟尿嘧啶缓释微球的制备及其体外释放的研究

目的：研究装载于不同分子量的PLGA中的5-氟尿嘧啶微球的制备方法及其在体外条件下的缓释行为。方法：以水包油包固复乳法将5-氟尿嘧啶包裹在高分子聚乳酸-聚羟基乙酸共聚物（PLGA）中,形成缓释微球,考察其大小,外观,包封率等理化性质,以紫外分光光度法为检测方法研究其体外释放行为。结果：经扫描电子显微镜观察,所制备的微球形态完整,大小较均匀。具有一定得包封率和载药量,体外释放研究表明其处方1和处方2的缓释时间为8天和23天。结论：以水包油包固复乳法制备的PLGA 5-氟尿嘧啶微球能够达到缓释的目的。     

Pharmacologically active microcarriers influence VEGF‐A effects on mesenchymal stem cell survival

Resistance of transplanted mesenchymal stem cells (MSCs) in post‐ischemic heart is limited by their poor vitality. Vascular‐endothelial‐growth‐factor‐A (VEGF‐A) as such or slowly released by fibronectin‐coated pharmacologically‐active‐microcarriers (FN‐PAM‐VEGF) could differently affect survival kinases and anti‐apoptotic mediator (e.g. Bcl‐2). Therefore VEGF‐A or FN‐PAM‐VEGF could differently enhance cell proliferation, and/or resistance to hypoxia/reoxygenation (H/R) of MSCs. To test these hypotheses MSCs were incubated for 6‐days with VEGF‐A alone or with FN‐PAM‐VEGF. In addition, MSCs pre‐treated for 24‐hrs with VEGF‐A or FN‐PAM‐VEGF were subsequently exposed to H/R (72‐hrs 3% O₂ and 3‐hrs of reoxygenation). Cell‐proliferation and post‐hypoxic vitality were determined. Kinases were studied at 30‐min., 1‐ and 3‐days of treatment. Cell‐proliferation increased about twofold (P < 0.01) 6‐days after VEGF‐A treatment, but by a lesser extent (55% increase) with FN‐PAM‐VEGF (P < 0.05). While MSC pre‐treatment with VEGF‐A confirmed cell‐proliferation, pre‐treatment with FN‐PAM‐VEGF protected MSCs against H/R. In the early phase of treatments, VEGF‐A increased phospho‐Akt, phospho‐ERK‐1/2 and phospho‐PKCε compared to the untreated cells or FN‐PAM‐VEGF. Afterword, kinase phosphorylations were higher with VGEF, except for ERK‐1/2, which was similarly increased by both treatments at 3 days. Only FN‐PAM‐VEGF significantly increased Bcl‐2 levels. After H/R, lactate dehydrogenase release and cleaved Caspase‐3 levels were mainly reduced by FN‐PAM‐VEGF. While VEGF‐A enhances MSC proliferation in normoxia, FN‐PAM‐VEGF mainly hampers post‐hypoxic MSC death. These different effects underscore the necessity of approaches suited to the various conditions. The use of FN‐PAM‐VEGF could be considered as a novel approach for enhancing MSC survival and regeneration in hostile environment of post‐ischemic tissues.     

基于魔芋葡甘聚糖微球的胶原覆层型微载体的制备研究

以魔芋葡甘聚糖(KGM)微球为基质,用1,4-丁二醇二缩水甘油醚将KGM微球进行活化,将胶原覆层到微球上,对胶原覆层进行再次交联,得到覆层均匀、稳定的微载体.通过四因素三水平的正交回归组合试验设计,考察了活化时间、蛋白质用量、偶联时间、交联剂用量对微载体细胞培养效果的影响.以Vero细胞培养效果为指标,制备胶原包被微载体的最佳工艺为活化时间5h、蛋白用量1∶0.1(球:蛋白)、偶联时间5h、交联剂用量每1gKGM加入0.5ml交联剂.在最优制备条件下,培养Vero细胞最大细胞密度可达到1.7×106 cells/ml,证明了胶原覆层的KGM微球作为动物细胞培养的微载体具有可行性.     

Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure.     

A New Spinel‐Layered Li‐Rich Microsphere as a High‐Rate Cathode Material for Li‐Ion Batteries

Li‐rich layered materials are considered to be the promising low‐cost cathodes for lithium‐ion batteries but they suffer from poor rate capability despite of efforts toward surface coating or foreign dopings. Here, spinel‐layered Li‐rich Li‐Mn‐Co‐O microspheres are reported as a new high‐rate cathode material for Li‐ion batteries. The synthetic procedure is relatively simple, involving the formation of uniform carbonate precursor under solvothermal conditions and its subsequent transformation to an assembled microsphere that integrates a spinel‐like component with a layered component by a heat treatment. When calcined at 700 °C, the amount of transition metal Mn and Co in the Li‐Mn‐Co‐O microspheres maintained is similar to at 800 °C, while the structures of constituent particles partially transform from 2D to 3D channels. As a consequence, when tested as a cathode for lithium‐ion batteries, the spinel‐layered Li‐rich Li‐Mn‐Co‐O microspheres obtained at 700 °C show a maximum discharge capacity of 185.1 mA h g^?1 at a very high current density of 1200 mA g^?1 between 2.0 and 4.6 V. Such a capacity is among the highest reported to date at high charge‐discharge rates. Therefore, the present spinel‐layered Li‐rich Li‐Mn‐Co‐O microspheres represent an attractive alternative to high‐rate electrode materials for lithium‐ion batteries.