PAMAM包覆脂质体的制备、表征及作为眼部递药载体的评价

制备了树枝状聚合物聚酰胺-胺2代和3代(PAMAM G2, PAMAM G3)包覆的葛根素(Puerarin, PUE)脂质体, 考察了脂质体包覆前后的粒径、Zeta电位的变化及包覆率和体外释放特性. 用异硫氰酸荧光素(FITC)标记PAMAM, 采用透射电镜和激光扫描共聚焦显微镜分别观察了PAMAM包覆脂质体和FITC-PAMAM包覆脂质体的形态. 采用改进的Valia-Chien扩散池及兔离体角膜评价了脂质体包覆前后角膜的药物渗透特性, 分别考察了脂质体包覆前后的角膜前滞留时间、角膜残留药量和角膜水化值. 研究结果表明, 包覆后的脂质体粒径略有增加, 但没有显著差异, Zeta电位由负变正, 并且随PAMAM比例的增加而增加. 透射电镜和激光扫描共聚焦显微镜观察结果显示, PAMAM能较好地包覆于脂质体表面. PAMAM G2的包覆率明显比PAMAM G3高. 包覆前后的脂质体释药特性相似, 均具有明显的缓释作用. PAMAM包覆PUE脂质体后, 与PUE水溶液和未包覆PUE脂质体相比, 其PUE离体兔角膜表观渗透系数、角膜前滞留时间及角膜残留药量均明显增加, 并具有显著差异, 其中PAMAM G3包覆脂质体优于PAMAM G2包覆脂质体. 水化值检测结果表明, PAMAM包覆PUE脂质体对角膜的刺激性不明显.     

激光扫描共聚焦显微技术在高分子科学研究中的应用

激光扫描共聚焦显微技术作为一种深层形态结构分析的重要研究方法,避免了繁琐的实验操作以及对样品的破坏,可直接给出材料内部结构的三维图像,在生物学、医学、材料科学和冶金学等研究领域都有广泛的应用.本文阐述了激光扫描共聚焦显微镜的工作原理及技术优势,综述了激光扫描共聚焦显微技术在高分子科学研究领域的应用进展,包括聚合物多组分...     

基于异硫氰酸荧光素标记溶菌酶和荷正电纳米金构建荧光共振能量转移平台检测广谱细菌

通过异硫氰酸荧光素标记的溶菌酶(FITC-Lys)和聚乙烯亚胺修饰的荷正电纳米金粒子(PEI-AuNPs)与细菌结合,构建了一个基于荧光共振能量转移(FRET)的平台用于广谱细菌检测.待检样本中存在细菌时,荷正电的FITC-Lys(能量供体)通过与细胞壁肽聚糖的识别作用和静电作用与细菌结合,荷正电的PEI-AuNPs(...     

异硫氰酸荧光纳米复合粒子的研制与应用

采用改进的St ber法制备了二氧化硅外壳的纳米复合荧光粒子。利用异硫氰酸荧光素(FITC)与3-氨基丙基三乙氧基硅烷(APTEOS)反应制备前驱体,再用正硅酸乙酯(TEOS)在一定的条件下水解与缩合,制备有机-无机纳米复合荧光颗粒,利用透射电子显微镜(TEM)测试表明,此纳米复合颗粒呈球形、大小均一,直径约为70 nm。制备的纳米复合荧光粒子经过多次水洗后,仍有较强的荧光特性,有效地防止FITC泄露。用激光共聚焦显微镜观测纳米复合荧光粒子标记的牛血清白蛋白(BSA),可以明显看出BSA上的绿色荧光。     

温度和pH双重敏感荧光复合微凝胶制备和表征

通过反相悬浮聚合法制备N-异丙基丙烯酰胺(NIPAM)和甲基丙烯酸(MAA)的共聚微凝胶P(NIPAM-co-MAA), 以其为模板, 利用3-氨丙基三乙氧基硅烷(APTES)在碱性条件下的水解缩合反应, 制备得到了由氨基修饰的P(NIPAM-co- MAA)/SiO₂高分子/无机复合微凝胶, 再通过异硫氰酸荧光素(FITC)与氨基的键和作用, 得到了具有核-壳结构的温度和pH双重敏感荧光复合微凝胶. 通过扫描电子显微镜(SEM)、傅立叶变换红外光谱(FT-IR)、热台偏光显微镜(POM)和共聚焦激光扫描显微镜(CLSM)等手段对复合微凝胶进行了结构和性质表征, 结果表明, 该复合微凝胶对温度和pH均具有良好的响应特性, 并在可见光激发下发出荧光.     

激光扫描共聚焦显微镜在光电材料中的应用

随着自然科学的发展,除了医学和生物研究领域,激光扫描共聚焦荧光显微镜(laser scanning confocal microscopy, LSCM)在材料科学研究中的作用越来越显现出来,特别在光电材料的研究中具有广泛的应用。结合多年的仪器管理和使用经验,以本学院的奥林巴斯激光扫描共聚焦荧光显微镜FV1000为例,对LSCM的成像特点、技术优势及其在光电材料成像中的应用情况进行了概述,以期能为LSCM在更多领域的应用和研究工作提供参考。     

激光共聚焦显微技术在共定位应用中的常见问题

荧光共定位分析是当今生物显微成像中一个极为常见的技术。本文从以下4个方面详细介绍了使用Zeiss LSM880型激光扫描共聚焦显微镜(confocal laser scanning microscope,CLSM)进行共定位研究时常见的问题及解决方法:1.如何用共聚焦进行共定位实验;2.如何进行共定位分析;3.如何评价共定位;4.如何判定共定位结果。在共定位研究中,只要按照共定位实验的基本要求拍摄图像,并按照上述方法进行分析,便可得到准确可靠的共定位分析结果。     

FITC标记重组灵芝免疫调节蛋白(rLz-8)在NB4细胞中的动态定位

实验发现, 重组灵芝免疫调节蛋白(rLz-8)可直接杀伤人急性早幼粒细胞白血病细胞株NB4. 利用异硫氰酸荧光素(FITC)标记rLz-8, 其相关的活性实验结果和晶体结构分析都表明, FITC没有影响rLz-8已知生物学功能. 通过激光共聚焦显微镜观察FITC-rLz-8在NB4细胞内的动态过程发现, FITC-rLz-8可识别细胞膜上的受体, 并可进入细胞质, 并最终富集在细胞核区域内. Annexin V-FITC双染检测结果显示, rLz-8对NB4细胞杀伤作用的可能机制是对NB4细胞凋亡的诱导作用, 在一定浓度范围内, 剂量与凋亡诱导率成正相关. 因此rLz-8能够诱导肿瘤细胞NB4发生凋亡的亚细胞学机制可定位在细胞核上.     

不同分子量N-琥珀酰壳聚糖的制备及其与K562肿瘤细胞间亲和性的初步探讨

用化学降解法制备不同分子量的壳聚糖 ,以其为原料合成了系列N 琥珀酰壳聚糖 ,然后用异硫氰酸荧光素进行荧光标记 ,再与K5 6 2肿瘤细胞共孵育 ,通过流式细胞仪检测细胞的荧光强度来确定不同分子量N 琥珀酰壳聚糖与K5 6 2肿瘤细胞间亲和性的强弱 ,为靶向抗肿瘤药物载体的研究提供初步的参考 .结果表明N 琥珀酰壳聚糖和K5 6 2肿瘤细胞间有较强的亲和性 ,随着分子量的增加 ,其亲和性逐渐减弱 .     

碲化镉量子点自组装膜的构建及其对溶菌酶的界面传感

利用自组装膜(SAMs)技术在石英片表面构建了碲化镉量子点SAMs.考察了组装液浓度、组装时间和聚电解质组装层数等组装条件对膜发光性能的影响,并用紫外可见吸收光谱仪、荧光光谱仪、共聚焦荧光显微镜和原子力显微镜对其进行了表征.基于溶菌酶对该SAMs的荧光具有猝灭效应,建立了一种快速灵敏测定痕量溶菌酶的界面荧光分析法,线性...     

Relation between pore sizes of protein crystals and anisotropic solute diffusivities

The diffusion of a solute, fluorescein, into lysozyme protein crystals with different pore structures was investigated. To determine the diffusion coefficients, three-dimensional solute concentration fields acquired by confocal laser scanning microscopy (CLSM) during diffusion into the crystals were compared with the output of a time-dependent 3-D diffusion model. The diffusion process was found to be anisotropic, and the degree of anisotropy increased in the order: triclinic, tetragonal and orthorhombic crystal morphology. A linear correlation between the pore diffusion coefficients and the pore sizes was established. The maximum size of the solute, deduced from the established correlation of diffusion coefficients and pore size, was 0.73 +/- 0.06 nm, which was in the range of the average diameter of fluorescein (0.69 +/- 0.02 nm). This proves that size exclusion is the key mechanism for solute diffusion in protein crystals. Hence, the origin of solute diffusion anisotropy can be found in the packing of the protein molecules in the crystals, which determines the crystal pore organization.     

FSiNPs mediated improved double immunofluorescence staining for gastric cancer cells imaging

A fluorescent silica nanoparticles (FSiNPs) mediated double immunofluorescence staining technique has been proposed for MGC-803 gastric cancer cells imaging by confocal laser scanning microscopy. Anti-CEA antibody and anti-CK19 antibody which can be both bonded to MGC-803 gastric cancer cells were first conjugated to fluorescein isothiocyanate (FITC) doped fluorescent silica nanoparticles (FFSiNPs) and RuBPY doped fluorescent silica nanoparticles (RFSiNPs), respectively. The MGC-803 gastric cancer cells were incubated with the mixture of anti-CEA antibody-conjugated FFSiNPs and anti-CK19 antibody-conjugated RFSiNPs, and subsequently imaged using confocal laser scanning microscopy. With this method, the in vitro cultured MGC-803 gastric cancer cells lines were successfully doubled labeled and distinguished through antigen-antibody recognition, together with the green and red signal of FFSiNPs and RFSiNPs simultaneously obtained without crossreactivity by confocal laser scanning microscopy imaging. By comparison with the conventional double immunofluorescence staining using green-emitting and red-emitting dyes, the photostability of this proposed method for confocal laser scanning microscopy imaging has been greatly improved. Furthermore, the ex vivo imaging of primary MGC-803 gastric cancer cells samples came from the tumor tissues of mice bearing the MGC gastric cancer tumor xenografts by this method have also been explored. The results demonstrate that the method offers potential advantage of photostability for the confocal laser scanning microscopy imaging of MGC-803 gastric cancer cells, and is applicable to the imaging of primary MGC-803 gastric cancer cells from the tumor tissues.     

Quantification of binary diffusion in protein crystals

The use of confocal laser scanning microscopy for visualization and quantification of binary diffusion within anisotropic porous material is described here for the first time. The dynamics of adsorption profiles of dianionic fluorescein, zwitterionic rhodamine B, and their mixture in the cationic native orthorhombic lysozyme crystal were subsequently analyzed. All data could be described by a classical pore diffusion model. There was no change in the adsorption characteristics, but diffusion decreased with the introduction of a second solute in the solution. It was found that diffusion is determined by the combination of steric and electrostatic interactions,while adsorption is dependent on electrostatic and hydrophobic interactions. Thus, it was established that the outcome of binary transport depends on the solute, protein, and crystal characteristics.     

Synthesis and characterization of dually labeled Pickering-type stabilized polymer nanoparticles in a downscaled miniemulsion system

Dual fluorescently labeled polymer particles were prepared in a downscaled Pickering-type miniemulsion system. Stable dispersions were obtained and the size of the hybrid particles could be varied between ca. 180 and 430 nm. Silica nanoparticles were employed as sole emulsifier, which were labeled by a fluorescein dye (FITC) or (encapsulated) quantum dots, and the polymer core was labeled by a perylene derivative. Downscaling of the Pickering-type miniemulsion system is intriguing by itself as it allows the use of precious nanoparticles as emulsifiers. Here, silica particles with a fluorescent core and an overall diameter between 20 and 40 nm were prepared and employed as stabilizer. The dual excitation and emission of both dyes was tested by fluorescence measurements and confocal laser scanning microscopy (cLSM).     

Choice of different dyes to label tyrosine and nitrotyrosine

In this work, we will present some attempts to analyze tyrosine and nitrotyrosine using capillary electrophoresis and either UV-Visible detection or laser-induced fluorescence (LIF) detection. An argon ion (488 nm) laser is used for fluorescein isothiocyanate (FITC) and 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F). A near infrared (780 nm) laser is used for NIR 780 derivatives. The UV-Visible limit of detection is 2.5 microM whereas it is in the range of 30 nM for LIF detection.     

Raman spectroscopic investigation of carbon nanowalls

Two-dimensional carbon nanowalls (CNWs) were prepared by microwave plasma-enhanced chemical-vapor deposition and scanning electron microscopy was used to observe their morphologies. The Raman observations of different sample orientations and polarizations show that CNWs are well crystallized. Micro-Raman scattering measurements were also carried out with different excitation laser lines (325, 488, 514, 532, and 633 nm). The D band shows a very strong shift of 46.19 cm(-1)eV with excitation laser energy and this has been explained by the double resonance effect. The decreasing intensity ratios IDIG and ID'/IG with increasing laser excitation energy were detected and discussed.     

Gentle immobilization of nonionic polymersomes on solid substrates

Vesicles from Pluronic L121 (PEO5-PPO68-PEO5) triblock copolymers were first stabilized by a permanent interpenetrating polymer network and then gently immobilized onto a glass or mica surface. Fluorescence-labeled micrometer-sized vesicles were visualized with confocal laser scanning microscopy, and smaller sized capsules, around 100 nm, were probed by liquid atomic force microscopy. The immobilized vesicles were weakly attached to a negatively charged surface via negatively charged polyelectrolytes in combination with Mg2+ ions and can be reversibly detached from the surface by slightly elevated temperatures. To illustrate that the immobilized vesicles remain responsive to external stimuli, we show that it is possible to transform their shape from spherical to cylindrical by introducing a second Pluronic, namely, P123 (PEO20-PPO70-PEO20). The detailed transition process has been recorded in real time by confocal laser scanning microscopy. Electron microscopy studies confirmed that a similar morphology change also occurs in the bulk.     

Synthesis and Characterization of ZnO Nanowires

Zinc oxide is a wide bandgap (3.37 eV) semiconductor with a hexagonal wurtzite crystal structure. ZnO prepared in nanowire form may be used as a nanosized ultraviolet light-emitting source. In this study, ZnO nanowires were prepared by vapor-phase transport of Zn vapor onto gold-coated silicon substrates in a tube furnace heated to 900 ?C. Gold serves as a catalyst to capture Zn vapor during nanowire growth. Size control of ZnO nanowires has been achieved by varying the gold film thickness…     

Investigation of the first shot phenomenon in MALDI mass spectrometry of protein complexes

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of noncovalent protein complexes is difficult, due to the disruptive nature of processes occurring during MALDI sample preparation and ion formation. Sometimes the observation of intact noncovalent protein complexes with MALDI is only possible if data are acquired from the first laser shot fired at a fresh sample; this is called the 'first shot phenomenon'. To study the origin of the first shot phenomenon, we used MALDI-MS and confocal laser scanning microscopy (CLSM) to examine typical MALDI sample preparations with embedded protein complexes, labeled with fluorophores. Fluorescence energy transfer techniques allowed the differentiation between intact and dissociated protein complexes with CLSM. In cases where a first shot behavior was observed by MALDI-MS, it was found to be accompanied by localization of protein complexes at the exterior of the sample crystals. Segregation of the large protein complexes to the exterior and dissociation of the complexes in the crystal interior during sample crystallization can rationalize this observation.     

Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures

Techniques based on two-photon excitation (TPE) allow three-dimensional (3D) imaging in highly localized volumes, of the order of magnitude of a fraction of a femtolitre up to single-molecule detection. In TPE microscopy a fundamental advantage over conventional widefield or confocal 3D fluorescence microscopy is given by the use of infrared (IR) instead of ultraviolet (UV) radiation to excite those fluorophores requiring UV excitation, hence causing little damage to the specimen or to fluorescent molecules outside the volume of the TPE event and allowing a deeper penetration within the sample compared with conventional one-photon excitation of fluorescence. In our laboratory, within the framework of a national INFM project, we have realized a TPE fluorescence microscope, part of a multipurpose architecture also including lifetime imaging and fluorescence correlation spectroscopy modules. The core of the architecture is a mode-locked Ti:sapphire infrared pulsed laser pumped by a high-power (5 W, 532 nm) solid-state laser and coupled to an ultracompact scanning head. For the source we have measured a pulse width from 65 to 95 fs as a function of wavelength (690-830 nm). The scanning head allows conventional and two-photon confocal imaging. Point spread function measurements are reported with examples of applications to the study of biological systems.