Synthesis and characterization of gold glyconanoparticles functionalized with sugars of sweet Sorghum syrup

Gold glyconanoparticles were synthesized by a simple, rapid, and eco-friendly method by using sweet Sorghum syrup for application in biomedicine and biotechnology. The nanostructures of the prepared gold nanoparticles were confirmed by using UV-visible absorbance, TEM, SAED, FTIR, EDAX, XRD, and photoluminescence analyses. The formation of gold nanoparticles at both room and boiling temperatures and kinetics of the reaction were monitored by UV-visible spectroscopy and TEM studies. TEM analysis revealed that the obtained nanoparticles were mono-dispersed and spherical in shape with an average particle size of 7 nm. The size of the nanoparticles was influenced by the concentration of Sorghum syrup. The presence of elemental gold was confirmed by EDAX analysis. Based on the FTIR analysis, it was observed that the sugars present in the Sorghum syrup possibly acts as capping agents. The zeta potential analysis revealed that the glyconanoparticles were negatively charged with a potential of -25 mV. The XRD and SAED patterns also suggest that the nanoparticles were crystalline in nature and these particles were found to exhibit visible photoluminescence. Fructose and glucose present in sweet Sorghum syrup were demonstrated as responsible sugars for the reduction of gold ions, and sucrose stabilized the formed nanoparticles. The proposed mechanism for the formation and stabilization of gold glyconanoparticles is based on the phenomenon of "macromolecular crowding." This is the first report on the use of sweet Sorghum syrup for the green synthesis of gold glyconanoparticles at both room and boiling temperatures.     

Gold DNA-conjugates: ion specific self-assembly of gold nanoparticles via the dG-quartet

The Oxytricha telomere DNA hairpin 5'-d(G4T4G4) immobilized on 13 nm gold nanoparticles forms a supramolecular assembly via dGC-quartets, as determined by the color change and by SEM. The aggregation is ion-dependent and selective for sodium ions. K+ is less efficient while Li+ and Cs+ do not drive the aggregation. This work is the first effort exploring the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of gold nanoparticles.     

Structure and activity of apoferritin-stabilized gold nanoparticles

A simple method for synthesizing gold nanoparticles stabilized by horse spleen apoferritin (HSAF) is reported using NaBH(4) or 3-(N-morpholino)propanesulfonic acid (MOPS) as the reducing agent. AuCl(4)(-) reduction by NaBH(4) was complete within a few seconds, whereas reduction by MOPS was much slower; in all cases, protein was required during reduction to keep the gold particles in aqueous solution. Transmission electron microscopy (TEM) showed that the gold nanoparticles were associated with the outer surface of the protein. The average particle diameters were 3.6 and 15.4 nm for NaBH(4)-reduced and MOPS-reduced Au-HSAF, respectively. A 5-nm difference in the UV-Vis absorption maximum was observed for NaBH(4)-reduced (530 nm) and MOPS-reduced Au-HSAF (535 nm), which was attributed to the greater size and aggregation of the MOPS-reduced gold sample. NaBH(4)-reduced Au-HSAF was much more effective than MOPS-reduced Au-HSAF in catalyzing the reduction of 4-nitrophenol by NaBH(4), based on the greater accessibility of the NaBH(4)-reduced gold particle to the substrate. Rapid reduction of AuCl(4)(-) by NaBH(4) was determined to result in less surface passivation by the protein. Methods for studying ferritin-gold nanoparticle assemblies may be readily applied to other protein-metal colloid systems.     

Controlled release of PEG chain from gold nanorods: Targeted delivery to tumor

Gold nanorods exhibit strong absorbance of light in the near infrared region, which penetrates deeply into tissues. Since the absorbed light energy is converted into heat, gold nanorods are expected to act as a contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. To construct a gold nanorod targeted delivery system for tumor a peptide substrate for urokinase-type plasminogen activator (uPA), expressed specifically on malignant tumors, was inserted between the PEG chain and the surface of the gold nanorods. In other words, we constructed PEG–peptide-modified gold nanorods. After mixing the gold nanorods with uPA, the PEG chain was released from the surface of the gold and subsequently nanorod aggregation took place. The formation of the aggregation was monitored as a decrease in light absorption at 900 nm. Tumor homogenate induced a significant decrease in this absorption. Larger amount of the PEG–peptide-modified gold nanorods bound to cells expressing uPA in vitro compared with control gold nanorods, which had scrambled sequence of the peptide. The PEG–peptide-modified gold nanorods showed higher accumulation in tumor than the control after they were injected intravenously into tumor-bearing mice, however, the density of the peptide on the surface of the gold nanorods was a key factor of their biodistributions. This targeted delivery system, which responds to uPA activity, is expected to be a powerful tool for tumor bioimaging and photothermal tumor therapy.     

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.     

A nanoparticle-based model delivery system to guide the rational design of gene delivery to the liver. 1. Synthesis and characterization

Nonviral gene delivery systems are amenable to forming colloidal particles with a wide range of physicochemical properties that include size, surface charge, and density and type of ligand presented. However, it is not known how to best design these particles without having a set of physicochemical design constraints that have been optimized for the intended gene delivery application. Here, a nanoparticle-based model delivery system is developed that can mimic the surface properties of nonviral gene delivery particles, and this model system is used to define design constraints that should be applied to next generation gene delivery particles. As a test case, a well-defined nanoparticle-based system is developed to guide the rational design of gene delivery to hepatocytes in the liver. The synthetic scheme utilizes monodisperse polystyrene particles and provides for variation of mean particle size and particle size distribution through variation in reaction conditions. The nanoparticles are PEGylated to provide stability in serum and also incorporate targeting ligands, e.g., galactose, at tunable densities. Four nanoparticles are synthesized from uniformly sized polystyrene beads specifically for the purpose of identifying design constraints to guide next generation gene delivery to the liver. These four nanoparticles are Gal-50 and Gal-140, that are galactosylated 50 and 140 nm nanoparticles, and MeO-50 and MeO-140, that are methoxy-terminated 50 and 140 nm nanoparticles. All four particles have the same surface charge, and Gal-50 and Gal-140 have the same surface galactose density. The availability of galactose ligands to receptor binding is demonstrated here by agglutination with RCA120.     

Size- and coating-dependent uptake of polymer-coated gold nanoparticles in primary human dermal microvascular endothelial cells

A library-orientated approach is used to gain understanding of the interactions of well-defined nanoparticles with primary human endothelial cells, which are a key component of the vasculature. Fifteen sequentially modified gold nanoparticles (AuNPs) based on three different core sizes (18, 35, 65 nm) and five polymeric coatings were prepared. The synthetic methodology ensured homogeneity across each series of particles to allow sequential investigation of the chemical features on cellular interactions. The toxicity of these nanoparticles, their uptake behavior in primary human dermal microvascular endothelial cells (HDMECs), and quantification of uptake were all investigated. The results of our studies indicated that high concentrations of gold nanoparticles (250 μg/mL) were nontoxic and that the number of internalized nanoparticles was related to nanoparticle size and surface chemistry. In summary, the positive-charged ethanediamine-coated AuNPs were internalized to a greater extent than the negative- or neutral-charged AuNPs. Moreover, differences in the amounts of internalized AuNPs could be shown for the three neutral-charged AuNPs, whereas the uptake of hydroxypropylamine-coated particles was preferred compared with glucosamine-coated or PEGylated AuNPs. Hydroxypropylamine-coated AuNPs were found to be the most efficient neutral-charged particles in overcoming the endothelial cell barrier and entering the cell.     

Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts

Leaf extracts of two plants, Magnolia kobus and Diopyros kaki, were used for ecofriendly extracellular synthesis of metallic gold nanoparticles. Stable gold nanoparticles were formed by treating an aqueous HAuCl₄ solution using the plant leaf extracts as reducing agents. UV–visible spectroscopy was used for quantification of gold nanoparticle synthesis. Only a few minutes were required for >90% conversion to gold nanoparticles at a reaction temperature of 95 °C, suggesting reaction rates higher or comparable to those of nanoparticle synthesis by chemical methods. The synthesized gold nanoparticles were characterized with inductively coupled plasma spectrometry (ICP), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), and particle analysis using a particle analyzer. SEM and TEM images showed that a mixture of plate (triangles, pentagons, and hexagons) and spherical structures (size, 5–300 nm) were formed at lower temperatures and leaf broth concentrations, while smaller spherical shapes were obtained at higher temperatures and leaf broth concentrations.     

Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery

Kinetics, biodistribution, and histological studies were performed to evaluate the particle‐size effects on the distribution of 15 nm and 50 nm PEG‐coated colloidal gold (CG) particles and 160 nm silica/gold nanoshells (NSs) in rats and rabbits. The above nanoparticles (NPs) were used as a model because of their importance for current biomedical applications such as photothermal therapy, optical coherence tomography, and resonance‐scattering imaging. The dynamics of NPs circulation in vivo was evaluated after intravenous administration of 15 nm CG NPs to rabbit, and the maximal concentrations of gold were observed 15–30 min after injection. Rats were injected in the tail vein with PEG‐coated NPs (about 0.3 mg Au/kg rats). 24 h after injection, the accumulation of gold in different organs and blood was determined by atomic absorption spectroscopy. In accordance with the published reports, we observed 15 nm particles in all organs with rather smooth distribution over liver, spleen and blood. By contrast, the larger NSs were accumulated mainly in the liver and spleen. For rabbits, the biodistribution was similar (72 h after intravenous injection). We report also preliminary data on the light microscopy and TEM histological examination that allows evaluation of the changes in biotissues after gold NPs treatment. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Functional gold nanoparticles for studying the interaction of lectin with glycosyl complex on living cellular surfaces

In this study, lectin-conjugated gold nanoparticles (GNPs) were prepared by standard biotin-streptavidin chemistry. The lectin-conjugated GNPs can be used as an indicator for studying the interaction of lectin with glycosyl complex on living cellular surfaces due to the high affinity of the lectin with saccharides. The interactions of two well-known lectins (Ricinus communis agglutinin and concanavalin A) and three different cell lines (HeLa, 293, and 293T) were selected here to establish this assay. Highly binding affinity of R. communis agglutinin with cells was demonstrated by conventional microscopic and UV-visible spectroscopic studies. In addition, the binding process can be inhibited by galactose, giving further proof of the binding mechanism.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

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.     

Optimization of PEGylation Conditions for BSA Nanoparticles Using Response Surface Methodology

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.     

Colloidal Au replacement assay for highly sensitive quantification of low molecular weight analytes by surface plasmon resonance

A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Hyper-Rayleigh scattering of protein-modified gold nanoparticles

The nonlinear optical properties of protein-modified gold nanoparticles has been studied by the hyper-Rayleigh scattering (HRS) technique. HRS signals from the nanoparticles coated with goat-anti-human IgG have been obtained when pumped with a laser pulse with a wavelength of 1064 nm. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles. This can be explained by a noncentrosymmetric effect. It was also found that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added due to gold nanoparticle aggregation. Our experiment found that the HRS method could produce a measurable signal with 10 microg/ml antigen added, while the colorimetric method using UV spectrum detection required 100 microg/ml of added antigen. The results show that the HRS measurement of immunogold nanoparticles could become a potential immunoassay in determining small levels of antigen in aqueous samples.     

Association of gold nanorods in water solutions: influence of globular proteins

By absorption spectroscopy method optical properties of gold nanorods (10x38 nm) and their interaction with globular protein bovine hemoglobin and bovine serum albumin were investigated. Nanorods behavior was studied in water solution and in solution of 97 mM NaCl under ultrasound action during 90 min and results were then compared. In water solutions nanorods coagulation (aggregation) was observed with reduced optical density of the longitudinal plasmon band widening at lamda>800 nm. In NaCI solution absorption spectra evolution had complex character and was in some degree analogous to the result that was obtained for two-dimensional grids of gold nanoparticles when changing the distance between them. By interacting with serum albumin stabilization of colloid solution and dissociation of nanorods aggregates were observed.     

G4-DNA-coated gold nanoparticles: synthesis and assembly

Here, we describe the preparation of stable 15 nm gold nanoparticles (Au-NPs) coated with parallel-stranded G-quadruplexes (G4-DNA), comprising phosphorothioate residues on both sides of the DNA. Phosphorothioate residues located on the surface of the coated particles can anchor them to noncoated ones. Their incubation with more than 20-fold excess of 15 nm citrate-stabilized Au-NPs leads to the formation of flower-shaped structures comprising a central noncoated particle and five to six G-quadruplex-coated ones at the periphery, as revealed by TEM imaging analysis. The absorption band of the structures is shifted toward long wavelengths compared to individual particles not connected to each other. We show a strong dependence of plasmon coupling strength on the length of the DNA connecting Au-NPs.     

Lactose-installed poly(ethylene glycol)-poly(d,l-lactide) block copolymer micelles exhibit fast-rate binding and high affinity toward a protein bed simulating a cell surface. A surface plasmon resonance study

Lactose molecules were installed on the surface of poly(ethylene glycol)-poly(d,l-lactide) (PEG-PLA) block copolymer micelles in the scope of seeking specific recognition by cell surface receptors at hepatic sites. This, in turn, is expected to result in the formation of a complex displaying prolonged retention times and thus enhanced cellular internalization by receptor-mediated endocytosis. The so-obtained particles based on a block copolymer of molecular weight 9400 g/mol (4900/4500 g/mol for the PEG and PLA blocks, respectively) were found to have an average hydrodynamic diameter of 31.8 nm, as measured by dynamic light scattering. Further, the particle size distribution (micro(2)/Gamma(2)) was found to be lower than 0.08. Lactose-PEG-PLA micelles (Lac-micelles) were then injected over a gold surface containing Ricinus communis agglutinin lectins simulating the aforementioned glycoreceptors, and their interaction was studied by surface plasmon resonance. Then, a kinetic evaluation was carried out, by fitting the observed data mathematically. It appears that Lac-micelles bind in a multivalent manner to the lectin protein bed, which logically results in low dissociation constants. Micelles bearing a ligand density of 80% (Lac-micelles 80%: 80 lactose molecules per 100 copolymer chains) exhibit fast association phases (k(a1) = 3.2 x 10(4) M(-)(1) s(-)(1)), but also extremely slow dissociation phases (k(d1) = 1.3 x 10(-)(4) s(-)(1)). Recorded sensorgrams were fitted with a trivalent model, conveying a calculated equilibrium dissociation constant (K(D1) = k(d1)/k(a1)) of about 4 nM. The importance of cooperative binding was also assessed, by preparing Lac-micelles bearing different ligand densities, and by discussing the influence of the latter on kinetic constants. Interestingly enough, whereas Lac-micelles 80% bind in a trivalent manner to the protein bed, Lac-micelles 20% are still capable of forming bivalent complexes with the same protein bed (K(D1) = 1360 nM). Therefore, despite enhanced kinetic values brought about by a supplementary bond, lower ligand densities appear to be more effective on a molecular basis.