Label-free Raman spectroscopy for accessing intracellular anticancer drug release on gold nanoparticles

We investigated glutathione (GSH)-induced purine or pyrimidine anticancer drug release on gold nanoparticle (AuNP) surfaces by means of label-free Raman spectroscopy. GSH-triggered releases of 6-thioguanine (6TG), gemcitabine (GEM), acycloguanosine (ACY), and fadrozole (FAD) were examined in a comparative way by means of surface-enhanced Raman scattering (SERS). The GSH-induced dissociation constant of GEM (or ACY/FAD) from AuNPs was estimated to be larger by more than 38 times than that of 6TG from the kinetic relationship. Tripeptide control experiments were presented to check the turn-off Raman signalling mechanism. Dark-field microscopy (DFM) and transmission electron microscopy (TEM) indicated the intracellular AuNP loads. After their cellular uptake, GEM, ACY, and FAD would not show SERS intensities as strong as 6TG. This may be due to easier release of GEM, ACY, and FAD than 6TG by intracellular reducing species including GSH. We observed fairly strong SERS signals of GEM and 6TG in cell culture media solution. Our CCK-8 cytotoxicity assay data support that 6TG-AuNPs did not exhibit a substantial decrease in cell viability presumably due to strong binding. Label-free confocal Raman spectroscopy can be utilized as an effective tool to access intracellular anticancer drug release.     

DNA-membrane complexes,mitochondria and aging

The results of extensive in vitro studies of DNA-lipid complexes allowed us to propose a model for the structure of such complexes and their involvement in the formation of DNA-membrane complexes (DMC). DMC seem to form the basis for such cellular structures as Bayer's junctions and nucleoid of bacteria, the nuclear pores, annulate lamellae and nucleoid of eucaryotes. The role of DMC in gene expression is discussed.Numerical density of mitochondria during cell aging correlates with the density of bacteria in batch culture. It is concluded that aging is caused by the unlimited growth of mitochondria and their subsequent degradation. The role of DMC in mitochondrial DNA damage at aging is discussed. The way of increasing the life span by controlling the density of mitochondria in a cell volume is likewise discussed. DMC formed between any two intracellular membranes can serve the basis for the membrane continuum in a cell.     

A study on the sizes and concentrations of gold nanoparticles by spectra of absorption, resonance Rayleigh scattering and resonance non-linear scattering

Liquid phase gold nanoparticles with different diameters and colors can be prepared using sodium citrate reduction method by controlling the amounts of sodium citrate. The mean diameters of gold nanoparticles are measured by transmission electron microscope (TEM). Gold nanoparticles with different sizes have specific absorption spectra. When the diameters of nanoparticles is between 12 and 41 nm, the maximum absorption peaks locate at 520-530 nm and there are red shifts gradually with the increase of diameters of gold nanoparticles. And when the size of gold nanoparticle is constant, the absorbance is proportional to the concentration of gold. Obvious resonance Rayleigh scattering (RRS) and the resonance non-linear scattering such as second-order scattering (SOS) and frequency-doubling scattering (FDS) appear at the same time as well, and the maximum scattering peaks are located at 286 nm (RRS), 480 nm (SOS) and 310 nm (FDS), respectively. When the concentration of gold is constant, absorbance and the intensities of RRS, SOS and FDS (I(RRS), I(SOS) and I(FDS)) have linear relationships with the diameters of gold nanoparticles. When the diameter of gold nanoparticle is constant, the absorbance and I(RRS), I(SOS), I(FDS) are directly proportional to the concentrations of gold nanoparticles. Therefore, it is very useful for studying the liquid phase gold nanoparticles by investigating the absorption, RRS, SOS and FDS spectra.     

Resonance scattering spectroscopy of gold nanoparticle

The gold nanoparticles in diameter of 10-95 nm have been prepared by Frens procedure, all of which exhibit a resonance scattering peak at 580 nm. The mechanism of resonance scattering for gold nanoparticle has been considered according to the wave motion theory of nanoparticle in liquid. The principle of superamolecular interface energy band(SIEB) has been set up and utilized to explain the relationship between the diameter and colors for gold nanoparticle in liquid. A novel spectrophotometric ruler for the determination of the diameter has been proposed according to the relationship of the maximum absorption wavelength and diameter.     

Examining changes in cellular communication in neuroendocrine cells after noble metal nanoparticle exposure

As nanoparticles enjoy increasingly widespread use in commercial applications, the potential for unintentional exposure has become much more likely during any given day. Researchers in the field of nanotoxicity are working to determine the physicochemical nanoparticle properties that lead to toxicity in an effort to establish safe design rules. This work explores the effects of noble metal nanoparticle exposure in murine chromaffin cells, focusing on examining the effects of size and surface functionality (coating) in silver and gold, respectively. Carbon-fibre microelectrode amperometry was utilized to examine the effect of exposure on exocytosis function, at the single cell level, and provided new insights into the compromised functions of cells. Silver nanoparticles of varied size, between 15 and 60 nm diameter, were exposed to cells and found to alter the release kinetics of exocytosis for those cells exposed to the smallest examined size. Effects of gold were examined after modification with two commonly used 'bio-friendly' polymers, either heparin or poly (ethylene glycol), and gold nanoparticles were found to induce altered cellular adhesion or the number of chemical messenger molecules released, respectively. These results support the body of work suggesting that noble metal nanoparticles perturb exocytosis, typically altering the number of molecules and kinetics of release, and supports a direct disruption of the vesicle matrix by the nanoparticle. Overall, it is clear that various nanoparticle physicochemical properties, including size and surface coating, do modulate changes in cellular communication via exocytosis.     

Relating surface-enhanced Raman scattering signals of cells to gold nanoparticle aggregation as determined by LA-ICP-MS micromapping

The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.     

Detection of Intracellular Proteins and Biomarkers Using Modified Silica Nanoparticles and Flow Cytometry In vitro

In this paper, antibody-modified silica nanoparticles were successfully synthesized, and their average diameter was (109±9) nm. These particles were mixed with cell extracts to target a protein, then, an antibody labeled with fluorescein isothiocyanate(FITC) was added to form FITC-labeled nanoparticle complexes and the product was analyzed using flow cytometry. The results confirm that the intracellular proteins and biomarkers were precisely and sensitivity detected by the novel method in vitro. The FITC intensity of Akt antibody-conjugated particles was increased ≥ 10-fold compared with that of control samples in MCF-7 cells. Furthermore, it can also simultaneously measure several proteins when modified with different antibodies.     

Physical vapor deposition of one-dimensional nanoparticle arrays on graphite: seeding the electrodeposition of gold nanowires

One-dimensional (1D) ensembles of 2-15 nm diameter gold nanoparticles were prepared using physical vapor deposition (PVD) on highly oriented pyrolytic graphite (HOPG) basal plane surfaces. These 1D Au nanoparticle ensembles (NPEs) were prepared by depositing gold (0.2-0.6 nm/s) at an equivalent thickness of 3-4 nm onto HOPG surfaces at 670-690 K. Under these conditions, vapor-deposited gold nucleated selectively at the linear step edge defects present on these HOPG surfaces with virtually no nucleation of gold particles on terraces. The number density of 2-15 nm diameter gold particles at step edges was 30-40 microm-1. These 1D NPEs were up to a millimeter in length and organized into parallel arrays on the HOPG surface, following the organization of step edges. Surprisingly, the deposition of more gold by PVD did not lead to the formation of continuous gold nanowires at step edges under the range of sample temperature or deposition flux we have investigated. Instead, these 1D Au NPEs were used as nucleation templates for the preparation by electrodeposition of gold nanowires. The electrodeposition of gold occurred selectively on PVD gold nanoparticles over the potential range from 700-640 mV vs SCE, and after optimization of the electrodeposition parameters continuous gold nanowires as small as 80-90 nm in diameter and several micrometers in length were obtained.     

Effect of thermodiffusion on pH‐regulated surface charge properties of nanoparticle

Surface properties of nanoparticle are of high importance in the field of biotechnology, drug delivery and micro/nanofabrication. In this article, we developed a comprehensive theoretical model and subsequently solved that numerically to study the effect of thermodiffusion of ions on surface charge properties of nanoparticle. The theoretical study has been done considering silica nanoparticle for two aqueous solutions NaCl and KCl. The effect of solution pH in conjunction with nanoparticle temperature on surface charge density has been obtained for different salt concentrations (1, 10 and 100 mM) and nanoparticle size (diameter of 2 and 100 nm). It is observed from the results that with increasing temperature of the nanoparticle, the negative surface charge density gets higher due to increasing thermodiffusion effect. It is also found out that the magnitude of surface charge density is higher for KCl solution than NaCl solution under same condition which is attributed mostly due to less thermodiffusion of counterions for KCl than NaCl. Present study also shows that magnitude of surface charge density decreases with increasing nanoparticle size until it reaches a limiting value (called critical size) above which the effect of nanoparticle size on surface charge density is insignificant.     

Stable polydiacetylene/ZnO nanocomposites with two-steps reversible and irreversible thermochromism: the influence of strong surface anchoring

The influence of pH value on gold nanoparticle production in the presence of Pluronic stabilizers is systematically investigated. The reactions are studied as a function of pH and at fixed concentrations of the two reactants, HAuCl(4) and P123 block copolymer. Results indicate that the reaction pathway during the nanoparticle formation can be controlled by varying pH. The nanoparticles synthesized at pH=11.12 have an average diameter of 9.6 nm with a narrow size distribution, and the Pluronics are adsorbed on individual gold particle surfaces to form core-shell structures via hydrophobic interactions. The present work provides an economic way to improve the dispersion and stabilization of gold nanoparticles and throws further light on the understanding of gold nanoparticle production using block copolymers.     

富精氨酸多肽修饰的金纳米粒子跨膜传输

考察了富精氨酸多肽功能化的金纳米粒子作为载体对细胞外物质的跨膜传输行为. 通过生物素(Biotin)与亲和素(Streptavidin)的亲和反应将具有特定跨膜功能的富精氨酸RRRRRRRR(R₈)多肽分子连接到多肽CALNN修饰的金纳米粒子表面, 实现粒子的功能化. 以荧光素为模型化合物, 利用激光共聚焦显微镜观察了纳米粒子的输送过程. 实验结果表明, 富精氨酸多肽功能化的金纳米粒子可以作为一种低毒高效的跨膜输送载体.     

Ferrocenyl‐Coupled N‐Heterocyclic Carbene Complexes of Gold(I): A Successful Approach to Multinuclear Anticancer Drugs

Four gold(I) carbene complexes featuring 4‐ferrocenyl‐substituted imidazol‐2‐ylidene ligands were investigated for antiproliferative and antivascular properties. They were active against a panel of seven cancer cell lines, including multidrug‐resistant ones, with low micromolar or nanomolar IC₅₀ (72 h) values, according to their lipophilicity and cellular uptake. The delocalized lipophilic cationic complexes 8 and 10 acted by increasing the reactive oxygen species in two ways: through a genuine ferrocene effect and by inhibiting the thioredoxin reductase. Both complexes gave rise to a reorganization of the F‐actin cytoskeleton in endothelial and melanoma cells, associated with a G1 phase cell cycle arrest and a retarded cell migration. They proved antiangiogenic in tube formation assays with endothelial cells and vascular‐disruptive on real blood vessels in the chorioallantoic membrane of chicken eggs. Biscarbene complex 10 was also tolerated well by mice where it led to a volume reduction of xenograft tumors by up to 80 %.     

金纳米粒子-胱氨酸三维网状结构的形成及其光谱特性研究

用柠檬酸三钠还原法制备了水溶性金纳米粒子, 粒子的平均粒径为4.5 nm, 它与胱氨酸作用后, 胱氨酸利用双硫键在其表面成功地进行了自组装, 获得了金纳米粒子-胱氨酸的三维网状结构. 用紫外-可见光谱、光散射光谱、透射电子显微镜等手段对胱氨酸组装前后的金纳米粒子进行了表征. 结果显示, 粒子与粒子之间, 通过静电引力形成了离子键, 吸收光谱变化明显, 金纳米粒子特征吸收峰由组装前518 nm红移到670 nm, 溶液颜色也相应由酒红色变为蓝紫色, 求出了金纳米粒子-胱氨酸三维网状结构形成过程中胱氨酸的最佳量, 金与胱氨酸的物质的量比为1∶1. 对于4.5 nm的金纳米粒子, 只有14%左右的胱氨酸在金纳米粒子的表面进行了自组装, 而多余的86%的胱氨酸未与金纳米粒子作用; 其共振瑞利散射光谱具有潜在的应用价值. 该研究对以金纳米粒子为基础的新材料制备进行了有益的探索.     

Extinction coefficient of gold nanoparticles with different sizes and different capping ligands

Extinction coefficients of gold nanoparticles with core size ranging from approximately 4 to 40 nm were determined by high resolution transmission electron microscopy analysis and UV-vis absorption spectroscopic measurement. Three different types of gold nanoparticles were prepared and studied: citrate-stabilized nanoparticles in five different sizes; oleylamide-protected gold nanoparticles with a core diameter of 8 nm, and a decanethiol-protected nanoparticle with a diameter of around 4 nm. A linear relationship between the logarithms of extinction coefficients and core diameters of gold particles was found independent of the capping ligands on the particle surface and the solvents used to dissolve the nanoparticles. This linear relation may be used as a calibration curve to determine the concentration or average size of an unknown nanoparticle or nanoparticle-biomolecule conjugate sample.     

Polymer size and concentration effects on the size of gold nanoparticles capped by polymeric thiols

Gold nanoparticles stabilized by thiol-terminated poly(ethylene glycol) monomethyl ethers with molecular weights ranging from 350 to 2000 have been prepared at thiol-to-gold molar ratios ranging from 3:1 to 1:8. Particle size distributions have been constructed for these particles from transmission electron microscopy images of hundreds of particles for each variation in synthetic conditions. The mean diameters of these particles range from 1.5 to 3.2 nm, with a slight increase in particle size with decreasing thiol content; these particles are smaller than those prepared using alkanethiols at similar thiol-to-gold ratios. Particles prepared under thiol-poor conditions exhibit much greater polydispersity than those prepared under thiol-rich conditions and include numerically rare large-particle outliers that contain much of the gold in the sample. The mean diameters of the gold nanoparticles decrease slightly with increasing polymer weight, especially under thiol-rich conditions. A simple model is developed to predict the trends in nanoparticle diameter that would result were the polymer's steric bulk protecting the nanoparticles from additional growth the principal factor controlling nanoparticle size in this system. This model predicts a much stronger dependence on thiol concentration than has been experimentally observed and a dependence on polymer molecular weight opposite to that experimentally observed. This suggests that the polymers' steric bulk is not the principal reason that these polymers yield smaller nanoparticles than alkanethiols at similar thiol-to-gold ratios. It is instead proposed that polar polymers may yield small nanoparticles by accelerating particle nucleation via coordination between functional groups in the polymer and atomic gold.     

Gold Nanoparticle Modified Electrodes Show a Reduced Interference by Cu(II) in the Detection of As(III) Using Anodic Stripping Voltammetry

Interference by Cu(II) causes serious problems in the detection of As(III) using anodic stripping voltammetry at gold electrodes. The behavior of Cu(II) and As(III) were examined at both a gold macro electrode and two kinds of gold nanoparticle modified electrodes, one where gold particles are deposited on glassy carbon (GC) and the other where basal plane pyrolytic graphite (BPPG) is the substrate. The sensitivity of As(III) detection was higher on gold nanoparticle modified electrodes than those on a macro gold electrode by up to an order of magnitude. In addition, the stripping peak of As(III) was narrower and more symmetric on a gold nanoparticle‐modified GC electrode, leading to analytical data with a lower limit of detection. At a macro gold electrode, the peak currents of Cu(II) were higher than those on gold nanoparticle modified electrodes. Accordingly, through the use of gold nanoparticle modified electrodes, the effect of copper interference to the arsenic detection can be reduced.     

Phosphorescent heavy-metal complexes for bioimaging

The application of phosphorescent heavy-metal complexes with d(6), d(8) and d(10) electron configurations for bioimaging is a new and promising research field and has been attracting increasing interest. In this critical review, we systematically evaluate the advantages of phosphorescent heavy-metal complexes as bioimaging probes, including their photophysical properties, cytotoxicity and cellular uptake mechanisms. The progress of research into the use of phosphorescent heavy-metal complexes for staining different compartments of cells, monitoring intracellular functional species, providing targeted bioimaging, two-photon bioimaging, small-animal bioimaging, multimodal bioimaging and time-resolved bioimaging is summarized. In addition, several possible future directions in this field are also discussed (133 references).     

Analytical performance of molecular beacons on surface immobilized gold nanoparticles of varying size and density

The high quenching efficiency of metal nanoparticles has facilitated its use as quenchers in molecular beacons. To optimize this system, a good understanding of the many factors that influence molecular beacon performance is required. In this study, molecular beacon performance was evaluated as a function of gold nanoparticle size and its immobilization characteristics. Gold nanoparticles of 4 nm, 15 nm and 87 nm diameter, were immobilized onto glass slides. Each size regime offered distinctive optical properties for fluorescence quenching of molecular dyes that were conjugated to oligonucleotides that were immobilized to the gold nanoparticles. Rigid double stranded DNA was used as a model to place fluorophores at different distances from the gold nanoparticles. The effect of particle size and also the immobilization density of nanoparticles was evaluated. The 4 nm and 87 nm gold nanoparticles offered the highest sensitivity in terms of the change in fluorescence intensity as a function of distance (3-fold improvement for Cy5). The optical properties of the molecular fluorophore was of significance, with Cy5 offering higher contrast ratios than Cy3 due to the red-shifted emission spectrum relative to the plasmon peak. A high density of gold nanoparticles reduced contrast ratios, indicating preference for a monolayer of immobilized nanoparticles when considering analytical performance. Molecular beacon probes were then used in place of the double stranded oligonucleotides. There was a strong dependence of molecular beacon performance on the length of a linker used for attachment to the nanoparticle surface. The optimal optical performance was obtained with 4 nm gold nanoparticles that were immobilized as monolayers of low density (5.7 × 10¹¹ particles cm⁻²) on glass surfaces. These nanoparticle surfaces offered a 2-fold improvement in analytical performance of the molecular beacons when compared to other nanoparticle sizes investigated. The principles developed in this study would assist in the design of solid phase molecular beacons using gold nanoparticles.     

Inhibition of HIV fusion with multivalent gold nanoparticles

The design and synthesis of a multivalent gold nanoparticle therapeutic is presented. SDC-1721, a fragment of the potent HIV inhibitor TAK-779, was synthesized and conjugated to 2.0 nm diameter gold nanoparticles. Free SDC-1721 had no inhibitory effect on HIV infection; however, the (SDC-1721)-gold nanoparticle conjugates displayed activity comparable to that of TAK-779. This result suggests that multivalent presentation of small molecules on gold nanoparticle surfaces can convert inactive drugs into potent therapeutics.     

Rational Engineering of a Dynamic,Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy-driven DNA nanomachine with three-dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy-driven catalytic reaction of intramolecular toehold-mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye-labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction-based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity-induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.