Drug and gene delivery using gold nanoparticles

Monolayer-functionalized gold nanoparticles provide attractive vehicles for pharmaceutical delivery applications as a result of their size and the unique properties and release mechanisms imparted by their monolayer. This review provides examples of recent advances in the field of drug and gene delivery using gold nanoparticles.     

Production of ubiquinone-10 using bacteria

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.     

Aptamer biosensor for protein detection using gold nanoparticles

Combining gold nanoparticles (GNPs) as fluorescence quencher and aptamer as probe, we have developed protein biosensors by using DNA-modified GNPs. We examined how the experimental design, such as the type of interaction between DNA strands and GNPs, temperature, and microenvironment of aptamer, influences the recognition ability of the biosensor. Under our experimental conditions, the recognition of protein by the complex of dye-labeled DNA hybridized with aptamer that is immobilized on GNPs (Ap-Im-GNPs) shows the best character in protein detection.     

Magnetic immobilization of bacteria using iron oxide nanoparticles

Bacterial cell immobilization is a novel technique used in many areas of biosciences and biotechnology. Iron oxide nanoparticles have attracted much attention in bacterial cell immobilization due to their unique properties such as superparamagnetism, large surface area to volume ratio, biocompatibility and easy separation methodology. Adhesion is the basis behind many immobilization techniques and various types of interactions determine bacterial adhesion. Efficiency of bacterial cell immobilization using iron oxide nanoparticles (IONs) generally depends on the physicochemical properties of the IONs and surface properties of bacterial cells as well as environmental/culture conditions. Bacteria exhibit various metabolic responses upon interaction with IONs, and the potential applications of iron oxide nanoparticles in bacterial cell immobilization will be discussed in this work.     

Multimerized siRNA cross-linked by gold nanoparticles

In this study, siRNAs terminated with thiol groups were multimerized and cross-linked using ～5 nm gold nanoparticles (AuNPs) via Au-S chemisorption that can be intracellularly reduced. AuNPs immobilized with single-stranded antisense siRNA were assembled with those with single-stranded sense siRNA via complementary hybridization or assembled with those with single-stranded dimeric sense siRNA. The multimerized siRNA cross-linked by AuNPs showed increased charge density and enhanced enzymatic stability, and exhibited good complexation behaviors with a polycationic carrier, linear polyethylenimine (L-PEI). The resultant multi-siRNA/AuNPs/L-PEI polyelectrolyte complexes exhibited far greater gene silencing efficiencies of green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF) compared to naked siRNA complexes. They could also be visualized by micro-CT imaging. The results suggest that AuNP-mediated multimerization of siRNAs could be a rational approach to achieve both gene silencing and imaging at a target tissue simultaneously.     

Detection of protein-DNA interaction and regulation using gold nanoparticles

The interaction between protein and DNA is usually regulated by a third species, an effector, which can be either a protein or a small molecule. Convenient methods capable of detecting protein-DNA interaction and its regulation are highly desirable research tools. In the current study, we developed a method to directly “visualize” the interaction between a protein-DNA pair and its effector through the coupling with gold nanoparticles (AuNPs). As a proof-of-concept experiment, we constructed a model system based on the interaction between the lac repressor (protein) and operator (DNA) and its interplay with the lac operon inducer isopropyl β-d-1-thiogalactopyranoside (IPTG, which inhibits the interaction between the lac repressor and operator). We coated AuNPs with the lac operator sequences and mixed them with the lac repressor. Because the lac repressor homotetramer contains two DNA binding modules, it bridged the particles and caused them to aggregate. We demonstrated that the assembly of DNA-modified AuNPs correlated with the presence of the corresponding protein and effector in a concentration-dependent manner. This AuNP-based platform has the potential to be generalized in the creation of reporter and detection systems for other interacting protein-DNA pairs and their effectors.     

Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles

Gold nanoparticles (AuNPs) were used as colorimetric probe and fluorescence quencher for affinity analysis of DNA aptamers toward their target mucin 1 (MUC1) peptide. Single-stranded DNA (ssDNA) aptamer-coated AuNPs showed increased stability (i.e., more resistant to aggregation induced by NaCl) in the presence of their target peptide due to the increase in steric protection conferred by the ssDNA-peptide complexes formed on the AuNPs. Based on changes in the UV-vis extinction spectrum of AuNPs (a measure of AuNPs aggregation) and fluorescence restoration of CY5-ssDNA upon ssDNA-peptide complex formation, the formation of the complexes and ssDNA sequence-dependent dissociation constant (K(d)) were determined. Besides the UV-vis and fluorescence measurements, the hydrodynamic diameters, zeta potential measurements, and transmission electron microscopy (TEM) images of AuNPs after various coatings supported the assay principle. The methodology presented herein provides a rapid and sensitive alternative solution for the identification of high affinity binders from systematic evolution of ligands by exponential enrichment (SELEX).     

Preparation of gold nanopowders and nanoparticles using chitosan suspensions

Simple methods for preparation of gold nanopowders and nanoparticles are reported. Gold/chitosan nanoparticles were prepared by using basic chitosan suspension as a dispersant and as a reductant. The resulting nanoparticles were processed by pyrolysis and thus obtain black gold nanopowder. The FESEM images indicate that most diameters of the nanopowder prepared were in the range of 50 and 200 nm. Hydrolysis is another quick decomposition method for chitosan. Acetic acid was adopted to implement the hydrolysis. The AEM images of the auberginic suspension show that the average gold nanoparticle diameter was less than 40 nm with good dispersion. Use of chitosan suspensions can produce gold nanopowder as well as gold nanoparticle without using toxic organic chemicals.     

Biosynthesis of gold nanoparticles by Azospirillum brasilense

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparticles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au⁺³ (HAuCl₄) to Au⁰. Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.     

Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold nanoparticles by using purified fungal phenol oxidases

The work shows the ability of cultured Basidiomycetes of different taxonomic groups—Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum, and Grifola frondosa—to recover gold, silver, selenium, and silicon, to elemental state with nanoparticles formation. It examines the effect of these metal and metalloid compounds on the parameters of growth and accumulation of biomass; the optimal cultivation conditions and concentrations of the studied ion-containing compounds for recovery of nanoparticles have been identified. Using the techniques of transmission electron microscopy, dynamic light scattering, X-ray fluorescence and X-ray phase analysis, the degrees of oxidation of the bioreduced elements, the ζ-potential of colloidal solutions uniformity, size, shape, and location of the nanoparticles in the culture fluid, as well as on the surface and the inside of filamentous hyphae have been determined. The study has found the part played by homogeneous chromatographically pure fungal phenol-oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) in the recovery mechanism with formation of electrostatically stabilized colloidal solutions. A hypothetical mechanism of gold(III) reduction from HAuCl₄ to gold(0) by phenol oxidases with gold nanoparticles formation of different shapes and sizes has been introduced.

     

Photothermal nanotherapeutics and nanodiagnostics for selective killing of bacteria targeted with gold nanoparticles

We describe a new method for selective laser killing of bacteria targeted with light-absorbing gold nanoparticles conjugated with specific antibodies. The multifunctional photothermal (PT) microscope/spectrometer provides a real-time assessment of this new therapeutic intervention. In this integrated system, strong laser-induced overheating effects accompanied by the bubble-formation phenomena around clustered gold nanoparticles are the main cause of bacterial damage. PT imaging and time-resolved monitoring of the integrated PT responses assessed these effects. Specifically, we used this technology for selective killing of the Gram-positive bacterium Staphylococcus aureus by targeting the bacterial surface using 10-, 20-, and 40-nm gold particles conjugated with anti-protein A antibodies. Labeled bacteria were irradiated with focused laser pulses (420-570 nm, 12 ns, 0.1-5 J/cm(2), 100 pulses), and laser-induced bacterial damage observed at different laser fluences and nanoparticle sizes was verified by optical transmission, electron microscopy, and conventional viability testing.     

Simple colorimetric sensing of trace bleomycin using unmodified gold nanoparticles

A simple colorimetric sensing platform for trace bleomycin (BLM) was proposed with the unmodified gold nanoparticles (AuNPs) as the sensing element. BLM has multiple N-donor functionality and exhibited strong coordination effect on AuNPs, which made it possible for the occurrence of ligand exchange of BLM with the weakly surface-bound citrate ions on AuNPs. Meanwhile, the positively charged BLM molecules further neutralized the surface charge, leading to increased van der Waals attractive force among AuNPs for rapid aggregation. This was reflected by the obvious color change from wine red to blue and rapid aggregation kinetics within 7.5 min. The BLM sensing based on unmodified AuNPs can be seen with the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for BLM was from 2 to 150 nM. The as-established colorimetric strategy opened a new avenue for trace BLM determination.     

Green synthesis of gold nanoparticles using Nyctanthes arbortristis flower extract

The present study explores the reducing and capping potentials of ethanolic flower extract of the plant Nyctanthes arbortristis for the synthesis of gold nanoparticles. The extract at different volume fractions were stirred with HAuCl₄ aqueous solution at 80 °C for 30 min. The UV–Vis spectroscopic analysis of the reaction products confirmed successful reduction of Au³⁺ ions to gold nanoparticles. Transmission electron microscope (TEM) revealed dominant spherical morphology of the gold nanoparticles with an average diameter of 19.8 ± 5.0 nm. X-ray diffraction (XRD) study confirmed crystalline nature of the synthesized particles. Fourier transform infra-red (FTIR) and nuclear magnetic resonance (NMR) analysis of the purified and lyophilized gold nanoparticles confirmed the surface adsorption of biomolecules during preparation and caused long-term (6 months) stability. Low reaction temperature (25 °C) favored anisotropy. The strong reducing power of the flower extract can also be tested in the green synthesis of other metallic nanoparticles.     

Development of neuraminidase detection using gold nanoparticles boron-doped diamond electrodes

Gold nanoparticles-modified boron-doped diamond (AuNPs–BDD) electrodes, which were prepared with a self-assembly deposition of AuNPs at amine-terminated boron-doped diamond, were examined for voltammetric detection of neuraminidase (NA). The detection method was performed based on the difference of electrochemical responses of zanamivir at gold surface before and after the reaction with NA in phosphate buffer solution (PBS, pH 5.5). A linear calibration curve for zanamivir in 0.1 M PBS in the absence of NA was achieved in the concentration range of 1 × 10⁻⁶ to 1 × 10⁻⁵ M (R² = 0.99) with an estimated limit of detection (LOD) of 2.29 × 10⁻⁶ M. Furthermore, using its reaction with 1.00 × 10⁻⁵ M zanamivir, a linear calibration curve of NA can be obtained in the concentration range of 0–12 mU (R² = 0.99) with an estimated LOD of 0.12 mU. High reproducibility was shown with a relative standard deviation (RSD) of 1.14% (n = 30). These performances could be maintained when the detection was performed in mucin matrix. Comparison performed using gold-modified BDD (Au–BDD) electrodes suggested that the good performance of the detection method is due to the stability of the gold particles position at the BDD surface.     

Naked eye detection of mutagenic DNA photodimers using gold nanoparticles

We developed a method to detect mutagenic DNA photodimers by the naked eye using gold nanoparticles. The stability of gold nanoparticles in a high ionic strength solution is maintained by straight ssDNA adsorbed physically on the AuNPs. However, we found that UV-irradiated DNA was less adsorptive onto gold nanoparticles because of a conformational change of UV-irradiated DNA. The accumulated deformation of the DNA structure by multiple-dimer formation triggered aggregation of the gold nanoparticles mixed with the UV-irradiated DNA and thus red to purple color changes of the mixture, which allowed colorimetric detection of the DNA photodimers by the naked eye. No fragmented mass and reactive oxygen species production under the UVB irradiation confirmed that the aggregation of gold nanoparticles was solely attributed to the accumulated deformation of the UV irradiated DNA. The degree of gold nanoparticles-aggregation was dependent on the UVB irradiated time and base compositions of the UV-irradiated oligonucleotides. In addition, we successfully demonstrated how to visually qualify the photosensitizing effect of chemical compounds in parallel within only 10 min by applying this new method. Since our method does not require any chemical or biochemical treatments or special instruments for purifying and qualifying the DNA photolesions, it should provide a feasible tool for the studies of the UV-induced mutagenic or carcinogenic DNA dimers and accelerate screening of a large number of drug candidates.     

CPMV-polyelectrolyte-templated gold nanoparticles

The use of polyelectrolyte surface-modified Cowpea mosaic virus (CPMV) for the templated synthesis of narrowly dispersed gold nanoparticles is described. The cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), is electrostatically bound to the external surface of the virus capsid; the polyelectrolyte promotes the adsorption of anionic gold complexes, which are then easily reduced, under mild conditions, to form a metallic gold coating. As expected, the templated gold nanoparticles can be further modified with thiol reagents. In contrast, reaction of polyelectrolyte-modified CPMV (CPMV-PA) with preformed gold nanoparticles results in the self-assembly of large, hexagonally packed, tessellated-spheres.     

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.     

Detection of cholesterol by digitonin conjugated gold nanoparticles

Functionalized colloidal gold is widely used for qualitative and quantitative detection of specific analytes. We report here a novel modification of gold nanoparticles by digitonin, a glycoside used for precipitating membrane cholesterol. The specific molecular recognition of cholesterol by digitonin gold nanoparticles (DGNP), make it an attractive alternative to the existing enzymatic methods for cholesterol sensing. To enable cholesterol binding, we modified mercapto modified GNPs with digitonin, by a simple esterification reaction. The blue shift in the plasmon absorption spectra of DGNP with different cholesterol concentrations accompanied by a decrease in the absorbance is the principle applied here for the estimation. The observed size reduction followed by cholesterol binding is reasoned due to the enhanced hydophobicity of the surface which in turn expels the water layers associated with the particles prior to cholesterol binding. The method exhibited linearity between concentration of cholesterol and the corresponding absorbance of the plasmon peak, in the range of 160-600 ng/mL with a detection limit of 100±9 ng/mL. Other steroids did not show any binding affinity towards DGNP. The method depicted here has potential for development as an enzyme free sensor for cholesterol although many factors need to be addressed to transform it for assaying samples like blood.