Organic acids and protein compounds causing the photoluminescence properties of natural rubber membranes and the quenching phenomena from Au nanoparticle incorporation

Natural rubber membranes were fabricated using latex from Hevea brasiliensis trees (clone RRIM 600) by casting, and controlling the time and temperature of thermal treatment. Three temperatures were used: 65, 80 and 120 °C and the corresponding annealing times of 6, 8, 10 and 12 h. The centrifugation of the latex produces the constituent phases: solid rubber (F1), serum or protein components (F2) and bottom fraction (F3). The photoluminescence properties could be correlated with organic acid components of latex. Natural rubber membranes were used as the active substrate (reducing agent) for the incorporation of colloidal Au nanoparticles synthesized by in situ reduction at different times. The intensity of photoluminescence bands assigned to the natural rubber decreases with the increase in amount of nanoparticles present on the membrane surface. It can be assumed that Au nanoparticles may be formed by reduction of the Au cation reacting with functional groups that are directly related to photoluminescence properties. However, the quenching of fluorescence may be attributed to the formation of a large amount of metal nanostructures on the natural rubber surface. Copyright © 2014 John Wiley & Sons, Ltd.     

Detection of hepatitis B surface antigen by target-induced aggregation monitored by dynamic light scattering

In the current work, a one-step, washing-free, homogeneous nanosensor assay has been constructed to sensitively detect hepatitis B surface antigen (HBsAg) based on the light scattering property of gold nanoparticles (GNPs) through a sandwich model. The two nanoprobes in this study were designed by conjugating monoclonal and polyclonal hepatitis B surface antibody (HBsAb) onto the GNPs of different diameters. First, the detection behavior of the combinations of different sizes of GNPs was evaluated and the optimized combination was determined. In analyzing HBsAg in Tris-HCl buffer, such bioassay composed of GNPs of approximately 50 and 100nm has a limit of detection (LOD) as high as 0.005IU/ml and a dose-dependent response ranging from 0.005 to 1IU/ml, which indicates its good diagnostic capability and provides a useful means to analyze protein biomarkers with low virus loads. Observation with transmission electron microscopy (TEM) provides direct evidence that the increase of hydrodynamic diameters resulted from the aggregation induced by immunological reactions. The bioassay also exhibits satisfactory specificity in analyzing HBsAg in serum media. Therefore, with its simple preparation, easy readout, and good stability, this bioassay has the potential to be developed into an automated and widely used biosensor assay.     

Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex

Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy–immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content.     

A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles

Background  

The synthesis of gold nanoparticles (GNPs) has received considerable attention with their potential applications in various life sciences related applications. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system, which has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. In the present study, we have demonstrated the synthesis of gold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines in India. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization via charge capping was investigated.     

细胞膜仿生修饰纳米粒肿瘤治疗的研究进展

大量研究证明,细胞膜仿生修饰通过将不同细胞膜包被于纳米粒表面,赋予纳米粒新的生物学功能.纳米粒被细胞膜仿生修饰后,获得了细胞膜表面丰富的蛋白质并保留了纳米粒的高载药能力,延长体内循环时间,使纳米粒具有逃避免疫系统,跨越各种生理屏障的能力.本文总结了近年来细胞膜仿生修饰纳米粒用于肿瘤治疗的最新进展,讨论了细胞膜仿生修饰纳...     

Leishmania chagasi: in vitro differentiation of promastigotes monitored by flow cytometry

A sequential development from a less infective to an infective stage of Leishmania promastigotes growing in culture has been previously reported. The aim of this work was to investigate whether freeze-fracture electron microscopy and flow cytometry would be able to provide some reliable morphological markers of in vitro differentiation of Leishmania chagasi promastigotes. The flow cytometry technique discriminates between the L. chagasi promastigotes from the different stages of their in vitro differentiation. The "forward scatter" intensity of the parasite, very high 15 hr after seeding when the parasites were very condensed and with a high DNA content per particle, strongly decreased during the culture course. Parallel experiments have shown a striking correlation between forward scatter intensity, growth curves, and infectivity of promastigote populations. By contrast, freeze-fracture techniques showed that in either less infective or infective promastigote plasma membranes, the intramembrane particles density in protoplasmic fracture faces (about 2800/micron 2) and in exoplasmic fracture faces (about 1000/micron 2) was independent of the time of cultivation. The amount of filipin lesions, which reflects the cholesterol content within the plasma membrane, was also constant throughout the culture course. Both data suggest that the architecture of the plasma membrane is an intrinsic characteristic of the promastigote stage. This study shows that whereas freeze-fracture electron microscopy does not provide markers for the differentiation of Leishmania promastigotes, flow cytometry may on the other hand be of value as a screening test for promastigote populations allowing the characterization of their developmental stages in in vitro cultures.     

Sensitive label-free electrochemical immunoassay based on a redox matrix of gold nanoparticles/Azure І/multi-wall carbon nanotubes composite

A sensitive label-free electrochemical immunosensing platform was designed by a redox matrix of gold nanoparticles (GNPs), Azure І and multi-wall carbon nanotubes (MWCNT) self-assemblying nanocomposite. To construct the immunosensor, MWCNT was first dispersed in Nafion (Nf) to obtain a homogeneous solution and then it was dropped on the surface of the gold electrode (Au). Then the positively-charged redox molecule, Azure І, was entrapped into MWCNT–Nf film to form a redox nanostructural membrane. Next, the negatively charged gold nanoparticles (GNPs) were assembled to the interface through the electrostatic force. Finally, carcinoembryonic antibody molecules could be absorbed into the GNPs/Azure І/MWCNT–Nf immobilization matrix. Using carcinoembryonic antigen (CEA) as a model protein, the electrochemical immunosensor exhibited good stability and reproducibility, as well as good selectivity and storage stability. This strategy presented a promising platform for sensitive and facile monitoring of CEA.     

Population changes in Leishmania chagasi promastigote developmental stages due to serial passage

Leishmania chagasi causes visceral leishmaniasis, a potentially fatal disease of humans. Within the sand fly vector, L. chagasi replicates as promastigotes which undergo complex changes in morphology as they progress from early stage procyclic promastigotes, to intermediate stage leptomonad and nectomonad promastigotes, and ultimately to terminal stage metacyclic promastigotes that are highly infective to vertebrates. This developmental progression is largely recapitulated in vitro using axenic promastigote cultures that have been passaged only a few times. Within a single passage (which takes about a week), axenic cultures progress from logarithmic to stationary growth phases; parasites within those growth phases progress from stages that do not have metacyclic cell properties to ones that do. Interestingly, repeated serial passage of promastigote cultures will result in cell populations that exhibit perturbations in developmental progression, in expression levels of surface macromolecules (major surface protease, MSP, and promastigote surface antigen, PSA), and in virulence properties, including resistance to serum lysis. Experiments were performed to determine whether there exists a direct relationship between promastigote developmental form and perturbations associated with repeated serial passage. Passage 2 to passage 4 L. chagasi cultures at stationary growth phase were predominately (>85%) comprised of metacyclic promastigotes and exhibited high resistance to serum lysis and high levels of MSP and PSA. Serial passaging 8, or more, times resulted in a stationary phase population that was largely (>85%) comprised of nectomonad promastigotes, almost completely devoid (<2%) of metacyclic promastigotes, and that exhibited low resistance to serum lysis and low levels of MSP and PSA. The study suggests that the loss of particular cell properties seen in cells from serially passaged cultures is principally due to a dramatic reduction in the proportion of metacyclic promastigotes. Additionally, the study suggests that serially passaged cultures may be a highly enriched source of nectomonad-stage promastigotes, a stage that has largely been characterized only in mixtures containing other promastigote forms.     

Immobilization of HIV-1 TAT peptide on gold nanoparticles: A feasible approach for siRNA delivery

RNA interference is one of the prosperous approaches for cancer treatment. However, small interfering RNA (siRNA) delivery to cancer cells has been faced with various challenges restricting their clinical application over the decades. Since ROR1 is an onco-embryonic gene overexpressed in many malignancies, suppression of ROR1 by siRNA can potentially fight cancer. Herein, a delivery system for ROR1 siRNA based on HIV-1 TAT peptide-capped gold nanoparticles (GNPs) was developed to treat breast cancer. Besides, we introduced a new feasible method for conjugating the peptide to the nanoparticles. Since the GNPs have high affinity to the sulfur, the findings demonstrated the peptide successfully conjugated to the nanoparticles via Au–S bonds. As positively charged nanoparticles showed high cellular uptake, we could use a low concentration of nanoparticles led to high efficient gene transfection with negligible cytotoxicity that was confirmed by flow cytometry, confocal microscopy, gel retardation, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Following transfection, downregulation of ROR1 and its targeted gene, CCND1, induced apoptosis in cancer cells. In conclusion, the reported capped GNPs could be potentially utilized for delivering negatively charged therapeutic agents in particular genes.     

Evaluation of size,morphology, concentration,and surface effect of gold nanoparticles on X-ray attenuation in computed tomography

Increasing attention has been focused on the use of nanostructures as contrast enhancement agents in medical imaging, especially in computed tomography (CT). To date, gold nanoparticles (GNPs) have been demonstrated to have great potential as contrast agents for CT imaging. This study was designed to evaluate any effect on X-ray attenuation that might result from employing GNPs with a variety of shapes, sizes, surface chemistries, and concentrations. Gold nanorods (GNRs) and spherical GNPs were synthesized for this application. X-ray attenuation was quantified by Hounsfield unit (HU) in CT. Our findings indicated that smaller spherical GNPs (13 nm) had higher X-ray attenuation than larger ones (60 nm) and GNRs with larger aspect ratio exhibited great effect on X-ray attenuation. Moreover, poly ethylene glycol (PEG) coating on GNRs declined X-ray attenuation as a result of limiting the aggregation of GNRs. We observed X-ray attenuation increased when mass concentration of GNPs was elevated. Overall, smaller spherical GNPs can be suggested as a better alternative to Omnipaque, a good contrast agent for CT imaging. This data can be also considered for the application of gold nanostructures in radiation dose enhancement where nanoparticles with high X-ray attenuation are applied.     

金纳米粒及其在药物传递系统中的应用研究进展

近年来,随着纳米材料科学的蓬勃发展,金纳米粒由于具有独特的光学和物理性质以及毒性小、比表面积大、表面可功能化修饰、易与药物分子结合等特点,其作为载体在药物传递系统中的应用已引起广泛关注。综述金纳米粒的特性、合成方法、体内分布与毒性以及在不同药物传递系统中的应用研究。     

A new concept in polymeric thin-film composite nanofiltration membranes with antibacterial properties

A new, thin film, biofouling resistant, nanofiltration (NF) membrane was fabricated with two key characteristics, viz. a low rate of silver (Ag) release and long-lasting antibacterial properties. In the new approach, nanoparticles were embedded completely in a polymeric thin-film layer. A comparison was made between the new thin-film composite (TFC), NF membrane and thin-film nanocomposite (TFN), and antibacterial NF membranes. Both types of NF membrane were fabricated by interfacial polymerization on a polysulphone sublayer using m-phenylenediamine and trimesoyl chloride as an amine monomer and an acid chloride monomer, respectively. Energy dispersive X-ray (EDX) microanalysis demonstrated the presence of Ag nanoparticles. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to study the cross-sectional and surface morphological properties of the NF membranes. Permeability and salt rejection were tested using a dead-end filtration cell. Ag leaching from the membranes was measured using inductively coupled mass spectrometry (ICP–MS). Morphological studies showed that the TFC NF membranes had better thin-film formation (a more compact structure and a smoother surface) than TFN NF membranes. Performance experiments on TFC NF membranes revealed that permeability was good, without sacrificing salt rejection. The antibacterial properties of the fabricated membranes were tested using the disk diffusion method and viable plate counts. The antibiofouling properties of the membranes were examined by measuring the quantity of bacterial cells released from the biofilm formed (as a function of the amount of biofilm present). A more sensitive surface was observed compared to that of a typical antibacterial NF membrane. The Ag leaching rates were low, which will likely result in long-lasting antibacterial and biofouling resistant properties.     

One-step method embedding superoxide dismutase and gold nanoparticles in silica sol-gel network in the presence of cysteine for construction of third-generation biosensor

A new and one-step method has been developed for the fabrication of superoxide dismutase (SOD) based biosensor. This method was used to form a silica sol-gel (SG) thin film and to immobilize SOD and gold nanoparticles (GNPs) in silica SG network for the fabrication of biosensor. The immobilized superoxide dismutase realized direct electron transfer between the enzyme and electrode surface, and the rate constants of the electrochemical process (ks) of SOD was markedly enhanced by GNPs. The electrochemical performance and influencing factors of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response to superoxide anion. The calibration range of superoxide anion was from 0.05 to 0.4 micromol L(-1). The proposed method exhibited the benefits of the advantages of self-assembly, nanoparticles and SG techniques. The fabrication of the SOD-modified electrode was easy and simple. The biosensor exhibited high sensitivity and long-term stability.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Multilayer associates based on oligonucleotides and gold nanoparticles

The development of nanodevices for the efficient transport of therapeutic molecules is one of the most urgent problems of modern molecular medicine. Noncovalent agents for the delivery of nucleic acids (NA) including those based on gold nanoparticles (GNPs) represent an attractive alternative to covalent systems, since it is easier in this case to provide the controlled release of NA. We have demonstrated the possibility to create potentially biocompatible associates of GNPs containing alternating layers of oligonucleotides and other polymers as a promising platform for the delivery of oligonucleotides into living cells. The multilayer (five layers) coated GNPs can be assembled by the sequential treatment of gold nanoparticles with nonthiolated oligonucleotide (ON), thiolated carboxyl-polyethylene glycol (SH-PEG3000-COOH), and linear polyethyleneimine (PEI). We have developed an algorithm for the analysis of multilayer coated GNPs by gel electrophoresis, photon correlation spectroscopy, and transmission electron microscopy. The assembly of associates bearing two oligonucleotide layers and having a net positive surface charge has been described. Multilayer coated GNPs were shown not to degrade in the presence of a high concentration of the major blood protein, serum albumin.     

金纳米粒在药物传递系统中的应用

金纳米粒是一种新型纳米载体,具有独特的理化、光学和生物学性质,且具有低毒性、低免疫原性、生物相容性好、体表面积大、易制备、粒径和形态可控、表面易修饰等优点,在生物医学领域和药物传递系统中具有广阔的应用前景。综述金纳米粒在小分子药物和基因药物传递系统中的应用研究新进展。     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.     

Detection of Vascular Endothelial Growth Factor Based on Gold Nanoparticles and Immunoreaction Using Resonance Light Scattering

In the study, a new assay of vascular endothelial growth factor (VEGF) has been developed by the use of gold nanoparticles (GNPs)–anti-VEGF conjugates. The immunoreaction between GNPs–anti-VEGF conjugates and VEGF took place in pH?7.5 PBS buffer solution after the addition of VEGF. The formation of GNPs modified VEGF immunocomplex resulted in the enhanced resonance light scattering (RLS) intensity at 388.0 nm. Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI _RLS) was proportional to the VEGF concentration in the range from 100 to 1,500 pg?mL^?1, with a detection limit of 60 pg?mL^?1. The surface plasma resonance absorption spectrum, the characteristics of RLS, the VEGF immunocomplex, and the optimum conditions of the immunoreaction have all been investigated. The VEGF concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay kit.