细胞对纳米材料的胞吞及其胞内定位相关机制的研究进展

纳米材料具有独特的理化性质,其在纳米生物医药技术中得到广泛的研究,有着良好的应用前景。纳米材料的尺寸分布在纳米级。使其入胞途径和转运方式与一般尺寸的物质略有不同。细胞可通过网格蛋白介导胞吞、陷窝小泡介导胞吞、吞噬作用和巨胞饮等胞吞方式摄取纳米颗粒。吞噬的方式及后续的转运和定位受细胞的类型、状态,以及纳米颗粒的理化性质如元素组成、尺寸、形状、电荷、表面修饰等多种因素共同影响。     

杂交鹅掌楸悬浮细胞高效摄取具有良好生物相容性的超微介孔氧化硅纳米颗粒

采用扫描电子显微镜(SEM)和激光共聚焦显微镜(LSCM)系统研究了超微介孔氧化硅纳米颗粒(MSNs)和杂交鹅掌楸悬浮细胞共孵育的互作特征.将MSNs通过异硫氰酸荧光素(FITC)标记后,再利用LSCM观察其被植物细胞摄取的情况.结果表明,5~15nm的MSNs可以通过内吞途径有效将FITC分子载入完整植物细胞.然而,在无MSNs作为载体的情况下,游离FITC分子则无法进入完整植物细胞.SEM观察结果进一步表明,MSNs容易聚集在完整植物细胞的表面,而且通过硅元素含量对比分析,直接证明了MSNs可以被完整植物细胞摄取到其内部.其次,利用荧光素二乙酸酯染料分析和MSNs共培养24h后的植物细胞的活性,其结果表明,这些细胞仍然保持很好的活力,而且也没有观察到明显的细胞死亡.最后发现,这些和MSNs共培养后的鹅掌楸细胞仍可以通过胚胎发育实现植株再生.综上所述,在植物生物学中,这种超微MSNs作为纳米载体可以应用到细胞的体外外源基因转染.     

水溶性纳米量子点在玉米幼根中运输的可视化

在纳米生物学效应研究中,纳米颗粒能否自由进入植物体内是一个亟待考证的关键问题。本文尝试采用一定浓度的水溶性纳米量子点溶液培养玉米幼根,然后徒手切片,用荧光显微镜直接观察发光量子点在根内的运输和分布。结果表明,量子点能够自由进入玉米根且在多种类型的细胞内都有分布;尤其是在导管内也观察到量子点,说明这种纳米材料能够穿透极厚的细胞壁输送到胞内。本文也讨论了该实验设计的优缺点。     

CdSe/ZnS-SA量子点细胞毒性的初步研究

目的：探讨链亲和素修饰的CdSe/ZnS核壳结构量子点（CdSe/ZnS-SA）对稳定转染pcDNA3.1/APP595/596质粒的人胚肾（HEK293）细胞的短期毒性作用.方法：将CdSe/ZnS-SA量子点与稳定转染pcD-NA3.1/APP595/596质粒的HEK293细胞共培育,在倒置荧光显微镜下观察细胞形态学变化;MTT法测定细胞活性;流式细胞术检测细胞凋亡率.结果：终浓度为2.5 nmol/L-20 nmol/L的CdSe/ZnS-SA量子点与HEK293细胞分别共育8h、16h、24 h后,细胞的形态无明显改变;终浓度为2.5 nmol/L-25 nmol/L的CdSe/ZnS-SA量子点与HEK293细胞分别共育8h、16 h、24 h,各处理组与对照组间,各处理组间的吸光度值、细胞凋亡率差异均无统计学意义（P＞0.05）.结论：一定浓度范围的CdSe/ZnS-SA量子点在短期内对稳定转染pcDNA3.1/APP595/596质粒的HEK293细胞无明显的毒性作用,具有较好的生物相容性.     

纳米粒子表面修饰对其与细胞膜相互作用的影响

纳米粒子在生物医学上的应用越来越广泛,其进入细胞的机制与规律是设计与开发的基础．已有研究发现,表面修饰不同亲疏水性基团的金纳米粒子,在内吞机制被抑制时,进入细胞的能力明显不同．更特别的是,粒子表面亲水性基团与疏水性基团呈间隔条纹规则排列的纳米粒子,与其他修饰成分相同仅排列不同的纳米粒子进入细胞的规律区别显著．这一特殊现象无法用已有的纳米粒子进入细胞的机制解释．本文针对该研究结果,将纳米粒子与细胞的体系简化,定量分析了3种不同纳米粒子进入细胞前后的不同状态,计算获得了表面修饰不同亲疏水性基团的纳米粒子与细胞膜之间相互作用的Flory．Huggins自由能．结果发现,修饰规则间隔排列亲疏水基团的纳米粒子,其作用自由能在与细胞接触前后变化最大．研究结果不仅解释了实验发现,同时预示了纳米粒子进入细胞的新机制．     

基于CdSe掺杂锐钛矿TiO_2的可见光体外灭活HL60细胞实验研究

用超声驱动方法合成了CdSe/TiO_2复合纳米粒子,并通过扫描电镜(SEM)、透射电镜(TEM)、X射线衍射(XRD)、紫外可见光(UV-Vis)吸收光谱和荧光(FS)光谱对CdSe/TiO_2复合纳米粒子进行表征。使用CCK-8法测定CdSe/TiO_2对白血病细胞的可见光光催化活性并通过SEM研究HL60细胞的表面超微结构形态。实验结果表明,用CdSe/TiO_2复合纳米粒子处理的组中观察到明显的HL60细胞生长抑制,并且HL60细胞在CdSe掺杂TiO_2复合纳米粒子作用下的PDT效率显著高于TiO_2,表明可以通过CdSe的修饰有效增强TiO_2的可见光光催化活性。此外,CdSe/TiO_2在可见光辐照下在4μg/m L的终值浓度下显示非常高的光动力效率,达76%。荧光光谱分析表明,CdSe/TiO_2复合纳米粒子可能通过分离光生空穴电子对提高此复合纳米粒子的光催化活性,从而提高CdSe/TiO_2对HL60细胞的PDT灭活效率。     

三种不同表面活性剂对羟基磷灰石纳米颗粒的表面修饰的比较

目的:本研究旨在通过不同方法修饰羟基磷灰石纳米颗粒并检测其稳定性及分散性。方法:首先采用水合热合成法制备羟基磷灰石纳米颗粒,然后用透射电镜(TEM)和场发射扫描电镜(SEM)对其表面形态结构进行表征。我们首次用溴化十六烷三甲基铵(CTAB),PEG2000和人血清对羟基磷灰石纳米颗粒通过共价结合或表面吸附的方式进行表面嫁接,并利用透射电镜,傅里叶红外光谱(FT-IR)和X射线衍射(XRD)对新合成的这三种纳米羟基磷灰石复合物的形貌,结构和晶粒粒径进行表征。对这三种羟基磷灰石纳米颗粒悬浮液的时间沉降曲线进行分析。在分散性上通过检测这三种羟基磷灰石复合物悬浮液在不同pH值下的Zeta电位并绘制Zeta-pH曲线。结果:我们发现CTAB修饰的羟基磷灰石纳米颗悬浮液的悬浮稳定性最佳,其次是PEG2000,最后是人血清。在pH=7.0时,CTAB修饰的羟基磷灰石纳米颗粒的zeta电位值是25.68 m V,而PEG2000修饰的Zeta电位是4.32m V,人血清修饰的Zeta电位是-13.23m V。结论:CTAB表面修饰的羟基磷灰石纳米颗粒相对于其它两种表面活性剂复合物具有更好的分散性和悬浮稳定性,与DNA/RNA结合能力更强。本课题的结果给羟基磷灰石纳米颗粒载体的应用提供了一种新的选择,有望利用亲和力更高的基因载体实现基因治疗,具有广阔的应用前景。     

细胞穿膜肽的研究进展

细胞穿膜肽是一类能携带大分子物质进入细胞的短肽,其穿膜能力不依赖经典的胞吞作用。经过对天然存在的细胞穿膜肽的生物化学性质研究,已经逐渐掌握了细胞穿膜肽的一些共有特性,这类物质均为带有正电荷的长短不等的多肽片段,其中富含精氨酸、赖氨酸等碱性氨基酸残基,二级结构皆具有α-螺旋的空间构象。利用这些特性,目前已人工合成了穿透力更强、效率更高的穿膜肽PEP-1、MPG,并且成功地携带大分子物质进入细胞发挥生物学活性。     

量子点细胞毒性及其作用机制

纳米粒子(NPS)在工业和研究中的使用急剧增加,因而这种材料面临一个其潜在毒性的问题。不幸的是,对纳米颗粒与纳米/生物界面可能发生的相互作用没有足够的了解。广大科技工作者正在积极寻求日益关注的纳米技术对人类的影响答案。我们将从NPS在生物媒体中的浓度,尺寸大小,电荷,和配位体的稳定性方面来了解纳米粒子的性质和他们在生物环境中对细胞毒所起的作用;并初步探讨已知的机制,量子点可以破坏细胞,包括氧化应激引起的活性氧(ROS)。微小浓度量子点足以造成长期持久的,甚至是跨代的影响。本文讨论了从纳摩尔到皮摩尔浓度的诱导细胞损伤的量子点(QDS)的浓度,这意味着含镉量子点可以发挥表观遗传毒性,纳米基因毒性,重金属基因的毒性。在此为评估包括量子点的在内的纳米毒性的的纳米材料,我们采用量子点作为一个例证,来阐述以科学为基础的发展到纳米毒理学的相关的问题。     

纳米材料在生物医学检测中的应用

纳米技术的兴起,对生物医学领域的变革产生了深远的影响。纳米材料是纳米技术发展的重要基础,它具有许多传统材料所不具备的独特的理化性质,因此在生物医学、传感器等重要技术领域有着广泛的应用前景。对几类常见的纳米材料包括纳米金、量子点、磁性纳米粒子、碳纳米管和硅纳米线在蛋白质、DNA、金属离子以及生物相关分子检测方面的应用进行综述。     

Fluid Phase Endocytic Uptake of Artificial Nano-Spheres and Fluorescent Quantum Dots by Sycamore Cultured Cells: Evidence for the Distribution of Solutes to Different Intracellular Compartments

Fluid phase endocytic uptake of external solutes in plant cells was further substantiated using artificial polystyrene nano-spheres (40 nm) and CdSe/ZnS quantum dots (20 nm). Both types of artificial nano-particles were taken up by sycamore-cultured cells. However, whereas polystyrene nano-spheres were delivered to the central vacuole, CdSe/ZnS nano-dots were sequestered into cytoplasmic vesicular structures. Using dextran-Texas Red (m.w. 3,000; d-TR) as additional marker, confocal micrographs confirmed the distinct topographic distribution of CdSe/ZnS quantum dots within the cell. Initially, d-TR and CdSe/ZnS quantum dots colocalized within cytoplasmic vesicles. After 18 h incubation, d-TR was distinctly localized in the vacuole whereas CdSe/ZnS quantum dots remained sequestered in cytoplasmic membranous compartments. The data provide a first evidence for the rapid distribution of solutes taken up by endocytosis to distinct intracellular compartments.Key Words: apoplast, assimilate partitioning, endocytosis, protoplasts, sucrose transport, sucrose uptake, vacuole     

Quenching of Fluorescence from CdSe/ZnS Nanocrystal QDs Near Copper Nanoparticles in Aqueous Solution

Significant quenching of fluorescence from CdSe/ZnS nanocrystal quantum dots (QDs) coated with mercaptoundecanoic ligands has been realized by copper nanoparticles (NPs). (a) Static quenching in the electrostatic association between the CdSe/ZnS QDs and cetyltrimethylammonium bromide-coated Cu NPs and (b) dynamic quenching of the same nanocrystals by polyvinylpyrrolidone-coated Cu NPs were studied. In both cases, the quenching of fluorescence from the CdSe/ZnS nanocrystals is sensitive to nanomolar concentrations of the copper NPs, and the quenching efficiency increases as spectral overlap between the CdSe/ZnS emission and the copper nanoparticle absorption increases. This suggests that the observed quenching is a result of energy transfer processes. These findings open new avenues for the utilization of Cu NPs in energy transfer-based applications.     

Energy transfer between CdSe/ZnS core/shell quantum dots and fluorescent proteins

Fluorescent proteins from the green fluorescent protein (GFP) family interact strongly with CdSe/ZnS quantum dots. Photoluminescence of GFP5 is suppressed by red-emitting CdSe/ZnS quantum dots with high efficiency in a pH-dependent manner. The elevated degree of quenching, around 90%, makes it difficult to analyze the remaining signal, and it is not clear yet whether FRET is the reason behind the quenching. When the donor is a green-emitting CdSe/ZnS quantum dot and the acceptor is the HcRed1 protein, it is possible to detect quenching of the donor and sensitized emission from the acceptor. It was verified that the sensitized emission has the low anisotropy characteristic of FRET. The present characterization identifies donor-acceptor pairs formed by fluorescent proteins and CdSe/ZnS quantum dots that are suitable for the exploration of cellular events. These donor-acceptor pairs take advantage of the exceptional photochemical properties of quantum dots allied with the unique ability of fluorescent proteins to act as gene-based fluorescent probes.     

Highly sensitive electrochemical detection of potential cytotoxicity of CdSe/ZnS quantum dots using neural cell chip

Cell chip was recently developed as a simple and highly sensitive tool for the toxicity assessment of various kinds of chemicals or nano-materials. Here, we report newly discovered potential cytotoxic effects of CdSe/ZnS quantum dots (QDs) on intracellular redox environment of neural cancer cells at very low concentrations which can be only detected by cell chip technology. Green (2.1 nm in diameter) and red (6.3 nm in diameter) QDs capped with cysteamine (CA) or thioglycolic acid (TA) were found to be toxic at 100 μg/mL when assessed by trypan blue and differential pulse voltammetry (DPV). However, in case of concentration-dependent cytotoxicity, toxic effects of TA-capped QDs on human neural cells were only measured by DPV method when conventional MTT assay did not show toxicity of TA-capped QDs at low concentrations (1-10 μg/mL). Red-TA QDs and Green-TA QDs were found to decrease electrochemical signals from cells at 10 μg/mL and 5 μg/mL, respectively, while cell viability decreased at 100 μg/mL and 50 μg/mL when assessed by MTT assay, respectively. The relative decreases of cell viability determined by MTT assay were 15% and 11.9% when cells were treated with 5-50 μg/mL of Red-TA QDs and 5-30 μg/mL of Green-TA QDs, respectively. However, DPV signals decreased 37.5% and 39.2% at the same concentration range, respectively. This means that redox environment of cells is more sensitive than other components and can be easily affected by CdSe/ZnS QDs even at low concentrations. Thus, our proposed neural cell chip can be applied to detect potential cytotoxicity of various kinds of molecular imaging agents simply and accurately.     

Quantum dots as a sensor for quantitative visualization of surface charges on single living cells with nano-scale resolution

We developed a technique using quantum dot (QD) as a sensor for quantitative visualization of the surface charge on biological cells with nano-scale resolution. The QD system was designed and synthesized using amino modified CdSe/ZnS nanoparticles. In a specially designed buffer solution, they are positively charged and can homogeneously disperse in the aqueous environment to label all the negative charges on the surfaces of living cells. Using a wide-field optical sectioning microscopy to achieve 2D/3D imaging of the QD-labeled cells, we determined the charge densities of different kinds of cells from normal to mutant ones. The information about the surface charge distribution is significant in evaluating the structure, function, biological behavior and even malignant transformation of cells.     

Analysis of Potential Radiosensitizing Materials for X-Ray-Induced Photodynamic Therapy

For a development of deep tumor treatment in photodynamic therapy, a feasibility of novel radiosensitizers induced by x-ray was investigated. The sensitizers are designed to generate reactive oxygen species (ROS) inside or outside the cell, possibly leading to damage exclusively on tumor cells and reservation of normal cells along the x-ray path. Taking note of the similarity in energy transfer mechanism in photocatalysts, scintillators, and particulate semiconductors, we chose TiO₂, ZnS:Ag, CeF₃, and quantum dots (CdTe and CdSe) in particulate form, which contain heavy atoms for efficient absorption of x-rays. A parameter study for x-ray operating conditions showed that in a typical scenario, photons with 20 to 170 keV energy are attenuated by 90% through the region of particle dispersed aqueous solution at varying concentration between 0.01 and 100 wt%. The amount of ROS generation under the exposure of polychromatic x-ray was measured using dihydroethidium reagent which detects an integrated amount of several species. Proportional increase in ROS generation to x-ray dose was observed for varying concentrations of TiO₂, ZnS:Ag, CeF₃, and CdSe quantum dot dispersions. Then, HeLa cells were mixed with aqueous solutions dispersed with sensitizing materials at a concentration of 3.0 mg/ml and were exposed to x-ray. Their survival fraction obtained by a cell proliferation reagent WST-1 immediately after the irradiation showed insignificant effects of sensitizing materials except at large doses. To enhance the sensitization effect, bio-conjugated CdSe quantum dots were internalized in the cytoplasm up to a concentration of 1.0 ng/ml. The cells were irradiated by x-ray up to 5 Gy, and their survival fraction was measured by the colony forming ability 9 days after irradiation. Survival fraction of the cells treated with quantum dots were less than those without quantum dots for all doses, suggesting that the colony forming ability is impaired by the internalized quantum dots.     

Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots

Chemical modification of the surface of CdSe/ZnS quantum dots (QDs) with small molecules or functional ligands often alters the characteristics of these particles. For instance, dopamine conjugation quenches the fluorescence of the QDs, which is a property that can be exploited for sensing applications if the conjugates are taken up into living cells. However, different sizes and/or preparations of mercaptocarboxylic acid solubilized QDs show very different properties when incubated with cells. It is unknown what physical parameters determine a QDs ability to interact with a cell surface, be endocytosed, escape from endosomes, and/or enter the nucleus. In this study, we examine the surface chemistry of QD-dopamine conjugates and present an optimized method for tracking the attachment of small biomolecules to the surface. It is found that the fluorescence intensity, surface charge, colloidal stability, and biological interactions of the QDs vary as a function of the density of dopamine on the surface. Successful targeting of QD-dopamine to dopamine receptor positive PC12 cells correlates with greater homogeneity of particle thiol layer, and a minimum number of ligands required for specific association can be estimated. These results will enable users to develop methods for screening QD conjugates for biological activity before proceeding to experiments with cell lines and animals.     

Metabolic alteration of Tetrahymena thermophila exposed to CdSe/ZnS quantum dots to respond to oxidative stress and lipid damage

CdSe/ZnS Quantum dots (QDs) are possibly released to surface water due to their extensive application. Based on their high reactivity, even small amounts of toxicant QDs will disturb water microbes and pose a risk to aquatic ecology. Here, we evaluated CdSe/ZnS QDs toxicity to Tetrahymena thermophila (T. thermophila), a model organism of the aquatic environment, and performed metabolomics experiments. Before the omics experiment was conducted, QDs were found to induce inhibition of cell proliferation, and reactive oxygen species (ROS) production along with Propidium iodide labeled cell membrane damage indicated oxidative stress stimulation. In addition, mitochondrial ultrastructure alteration of T. thermophila was also confirmed by Transmission Electron Microscope results after 48 h of exposure to QDs. Further results of metabolomics detection showed that 0.1 μg/mL QDs could disturb cell physiological and metabolic metabolism characterized by 18 significant metabolite changes, of which twelve metabolites improved and three decreased significantly compared to the control. Kyoto Encyclopedia of Genes and Genomes analysis showed that these metabolites were involved in the ATP-binding cassette transporter and purine metabolism pathways, both of which respond to ROS-induced cell membrane damage. In addition, purine metabolism weakness might also reflect mitochondrial dysfunction associated with energy metabolism and transport abnormalities. This research provides deep insight into the potential risks of quantum dots in aquatic ecosystems.     

Preparation and Biological Effect of Nucleotide-Capped CdSe/ZnS Quantum Dots on Tetrahymena thermophila

In this paper, we described the preparation and characterization of different types of modified CdSe/ZnS quantum dots (QDs) and explored the biological effects of QDs with different surface modifications on the whole growth of unicellular protozoan Tetrahymena thermophila BF(5) using a thermal activity monitor air isothermal microcalorimeter. Our results demonstrated that adenosine 5'-monophosphate (AMP) showed stronger interaction with QDs than other types of nucleotide. AMP-QDs could stimulate the growth of T. thermophila while mercaptoacetic acid-capped CdSe/ZnS quantum dots inhibited it. In addition, the population density determination and fluorescence imaging of T. thermophila BF(5) also confirmed the results obtained from microcalorimetry. It is believed that this approach will provide a more convenient methodology for the kinetics and thermodynamics of microorganism when coexisting with QDs in real time, and all of which are very significant to understanding the effect of QDs to organism.     

Examination of the stability of hydrophobic (CdSe)ZnS quantum dots in the digestive tract of rats.

Semiconductor quantum dots show promise as alternatives to organic dyes for biological labelling because of their bright and stable photoluminescence. The typical quantum dots is CdSe because colloidal synthesis for nanocrystals of this semiconductor is well established. CdSe is usually passivated with zinc sulfide. While the cytotoxicity of bulk CdSe is well documented, questions about (CdSe)ZnS potential toxicity and behaviour in vivo remain unanswered. The distribution and stability of (CdSe)ZnS quantum dots in Wistar line rats' digestive tract were investigated. Hydrophobic quantum dots were mixed with fat or sonificated in water and administered orally. The distribution and stability of quantum dots moving through the digestive system of rats was followed by fluorescence spectroscopy. In both ways prepared quantum dots were degraded in the digestive tract of animals. Quantum dots mixed with fat were more stable and degraded more slowly than quantum dots sonificated in water. The data obtained suggest possible toxicity of (CdSe)ZnS quantum dots due to the liberation of Cd(2+).