Vladimir A. Bogatyrev

On the Enhanced Antibacterial Activity of Antibiotics Mixed with Gold Nanoparticles.

The bacterial action of gentamicin and that of a mixture of gentamicin and 15-nm colloidal-gold particles onEscherichia coli K12 was examined by the agar-well-diffusion method, enumeration of colony-forming units, and turbidimetry. Addition of gentamicin to colloidal gold changed the gold color and extinction spectrum. Within the experimental errors, there were no significant differences in antibacterial activity between pure gentamicin and its mixture with gold nanoparticles (NPs). Atomic absorption spectroscopy showed that upon application of the gentamicin-particle mixture, there were no gold NPs in the zone of bacterial-growth suppression in agar. Yet, free NPs diffused into the agar. These facts are in conflict with the earlier findings indicating an enhancement of the bacterial activity of similar gentamicin–gold nanoparticle mixtures. The possible causes for these discrepancies are discussed, and the suggestion is made that a necessary condition for enhancement of antibacterial activity is the preparation of stable conjugates of NPs coated with the antibiotic molecules.

Analytical and Theranostic Applications of Gold Nanoparticles and Multifunctional Nanocomposites

Gold nanoparticles (GNPs) and GNP-based multifunctional nanocomposites are the subject of intensive studies and biomedical applications. This minireview summarizes our recent efforts in analytical and theranostic applications of engineered GNPs and nanocomposites by using plasmonic properties of GNPs and various optical techniques. Specifically, we consider analytical biosensing; visualization and bioimaging of bacterial, mammalian, and plant cells; photodynamic treatment of pathogenic bacteria; and photothermal therapy of xenografted tumors. In addition to recently published reports, we discuss new data on dot immunoassay diagnostics of mycobacteria, multiplexed immunoelectron microscopy analysis of Azospirillum brasilense, materno-embryonic transfer of GNPs in pregnant rats, and combined photodynamic and photothermal treatment of rat xenografted tumors with gold nanorods covered by a mesoporous silica shell doped with hematoporphyrin.

Gold nanorods: Synthesis and optical properties

The main results of studying the synthesis, growth mechanisms, and optical properties of gold nanorods published in the last 5–8 years are briefly reviewed. Hydrosols of gold nanorods with variable axial ratios are synthesized in the micellar solution of ionic surfactants by sead-mediated growth procedure using the stage of particle separation in the glycerol concentration gradient. Results of synthesis in systems containing one surfactant, albeit with different Ag/Au molar ratios and different amounts of gold seeding particles, agree with the published data. It is shown that, in the case of the mixture of two surfactants, the Ag/Au ratio is an efficient controlling parameter of the synthesis of nanorods with large axial ratios. The extinction and differential light scattering, spectra dynamic light scattering, and the depolarization of laser light scattering at 90° are used for the optical control of synthesis. Three fractions are observed in separated samples. One of these fractions is characterized by the only short wavelength plasmon resonance at 570 nm corresponding, in agreement with the published data, to cubic particles. Measurements of the extinction spectra of nanorods in water-glycerol mixtures reveal higher sensitivity of the longitudinal plasmon resonance to the dielectric environment relative to the transverse resonance. It is shown theoretically and experimentally that the relative shift of plasmon resonance is proportional to the relative increment of refractive index of the surrounding medium. To calculate optical properties of nanorods, we employed a model of cylinders with semispherical ends (s-cylinders) corresponding to the shape of real particles and admitting the exact solution by the T-matrix method with a computational burden that is an order of magnitude lower than that used in the discrete dipole method. The set of dependences of the longitudinal resonance wavelength on the axial ratio of different-thickness particles complies with our data and published measurements. Theoretical and experimental values of depolarization ratio IVH/IVV for nanorods and nanospheres with different sizes prepared with both citrate (15–46 nm) and original thiocyanate (90 nm) reduction of HAuCl4 are compared. It is shown that the depolarization parameter of light scattered by a nanorod suspension can exceed the theoretical limit (1/3) for common dielectric particles. The measured 10%-depolarization ratio for 90-nm spheres was far beyond the set of “size-depolarization” measurements for 15–46-nm-dia particles prepared by the citrate method and is indicative of the improved spherical morphology of 90-nm particles. This assumption was confirmed by TEM data, which also revealed both the presence of a noticeable amount of nanorods with a large axial ratio and “nanowires” of about the same thickness. A new analytic calibration for determining the diameter of spherical particles (5–100 nm) by the spectral position of the sol extinction maximum is proposed.

Preparation and optical scattering characterization of gold nanorods and their application to a dot-immunogold assay

We describe optical monitoring of the synthesis of gold nanorods (NRs) based on seed-mediated growth in the presence of the soft surfactant template cetyltrimethyilammonium bromide. To separate NRs from spheres and surfactants we fractionated samples in the density gradient of glycerol. The optical properties of NRs were characterized by extinction and differential light-scattering spectra (at 90 degrees, 450-800 nm) and by the depolarization light-scattering ratio, I(vh)/I(vv), measured at 90 degrees with a helium-neon laser. Theoretical spectra and the I(vh)/I(vv) ratios were calculated by the T-matrix method as applied to randomly oriented NRs, which were modeled by right-circular cylinders with semispherical ends. The simulated data were fitted to experimental observations by use of particle length and width as adjustable parameters, which were close to the data yielded by transmission electron microscopy. The sensitivity of the long-wavelength resonance of NRs to the dielectric surroundings was examined both experimentally and theoretically by comparison of the extinction spectra of NRs in water and in a 25% glycerol solution. Finally, we discuss the application of NR-protein A conjugates to a dot-immunogold assay with the example of biospecific staining of human IgG molecules adsorbed onto small membrane spots.

Optical Properties and Biomedical Applications of Nanostructures Based on Gold and Silver Bioconjugates

We discuss optical properties of single and aggregated colloidal gold and silver conjugates that can be fabricated by adsorption of a biopolymer onto nanoparticle surfaces. We start with a discussion of two-layer and multilayer optical models for colloidal gold and silver nanoparticle conjugates that consist of a metal core and a polymer shell formed by recognizing and target molecules. The point at issue is the core-size optimization of conjugate-based nanosensors as elementary transducers of molecular binding events into optical signals. We present a detailed discussion of optical properties of various aggregated conjugate-based structures such as bispheres, linear chains, plane arrays on a rectangular lattice, compact and porous clusters embedded on a cubic body-centerd lattice, and random fractal aggregates. Our attention is focused on the following topics: (1) statistical and orientation averaging of optical observables; (2) dependence of extinction and scattering spectra on the optical binary coupling of conjugates; (3) optical effects related to the chain-like structures; (4) effects of polymer coating, interparticle spacing, and cluster structure; (5) simulation of kinetic changes in the optical properties of aggregated sols formed during biospecific binding. Finally, we discuss experimental data and biomedical applications of metal nanoparticles and their biospecific conjugates in various biomedical studies.

Measurement of mean size and evaluation of polydispersity of gold nanoparticles from spectra of optical absorption and scattering

Two methods for determination of the mean size of gold nanoparticles, based on measurement of the wavelengths of the maxima λmax of side scattering and extinction in the range 400–700 nm, are compared. Four sols with mean particle diameters d of about 15, 20, 25, and 30 nm, measured using the dynamic light-scattering technique, were studied experimentally. The slope of the size dependence λmax(d) of the spectral position of the scattering peak exceeded that for the extinction peak by a factor of 2.4. This fact ensures a substantially higher accuracy of the scattering method. For simulating polydispersity, mixtures of three colloids with particle diameters of 20, 25, and 30 nm were used: sample S1, with a size distribution close to the normal one of around 25 nm, and sample S2, with equal concentrations of each of the components. The extinction spectra of mixtures S1 and S2 and the initial 25-nm sol (S0) were virtually identical, whereas their scattering spectra showed a pronounced increase in the peak amplitude in the series S0, S1, S2. These results agree with calculations based on the Mie theory. Thus, scattering spectra offer advantages over extinction spectra not only in measuring the mean size of gold particles but also in evaluating their polydispersity.

A Multilayer Model for Gold Nanoparticle Bioconjugates: Application to Study of Gelatin and Human IgG Adsorption Using Extinction and Light Scattering Spectra and the Dynamic Light Scattering Method

A new model of colloidal gold (CG) bioconjugates is proposed. The model consists of a gold core and a primary polymer shell formed during conjugate synthesis. Additionally, the conjugate includes a secondary shell formed during its interaction with target molecules. Each of the inhomogeneous shells is modeled by the arbitrary number of discrete layers. Using Mie theory for multilayered spheres, we calculated the extinction and static light scattering (SLS, at 90°) spectra, as well as differential spectra ΔA(λ), ΔI(λ) describing the effects of primary and secondary shells. Our calculations are performed for the conjugates with gold particle diameters d = 10–160 nm and two 5-nm shells. The primary shell consists of two 2.5-nm layers with the refractive indices of 1.50 and 1.45; the secondary shell, of two 2- and 3-nm layers with the refractive indices of 1.45 and 1.40. The differential spectra are related to the adsorption of target molecules and possess a characteristic resonance that is shifted to the red region of spectra compared to the usual localized plasmon resonances of gold particles. The maximal values of differential resonances ΔAmax and ΔImax are observed for gold particles with diameters about 40–60 nm (extinction spectra) or 70–90 nm (the SLS spectra). The adsorption of human gamma-globulin (hIgG) and gelatin onto 15- and 34-nm gold particles was studied using the SLS and extinction spectra in combination with the dynamic light scattering measurements. It is shown that the thickness of adsorbed layer is equal to 5–6 nm for hIgG and to 15–18 nm for gelatin. The experimental extinction and SLS spectra for CG + hIgG conjugates are well explained by a simple model with the gold core and homogeneous polymer coating. For the CG + gelatin conjugates, we used the new model with inhomogeneous polymer coating, which is modeled by 10 discrete layers with the total thickness of 16–18 nm and exponential spatial profile of shell refractive index.

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freunds adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freunds adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.

Quantitative cell bioimaging using gold-nanoshell conjugates and phage antibodies.

The authors describe a quantitative evaluation of the efficacy of cell labeling with plasmon-resonant light-scattering nanoparticles used as contrast agents for dark-field microscopy imaging. The experimental model is based on the biospecific labeling of pig embryo kidney (SPEV) cells with primary phage antibodies, followed by the dark-field microscopic visualization using conjugates of silica/gold nanoshells with secondary rabbit antiphage antibodies. To quantify nanoparticle binding, the authors introduce the labeling-efficacy factor (LEF) which is equal to the ratio of the bound-particle pixels per cell to the total number of pixels occupied by the cell. The LEF is calculated by an imaging-analysis algorithm based on the freely available ImageJ Java-based processing code. In terms of the LEF, a distinct difference was found between intact, nonspecifically labeled, and biospecifically labeled cells.

Spectra of resonance light scattering of gold nanoshells: Effects of polydispersity and limited electron free path

The effects of the polydispersity of the structure of gold nanoshells and of the limited electron free path in a thin metal layer on the spectra of resonance light scattering of a suspension of two-layer nanoparticles are studied theoretically and experimentally for the first time. It is shown theoretically that both factors lead to a broadening of the plasmon resonance in light scattering and to a change in its magnitude. To experimentally test the calculations, two samples of nanoshells based on gold and silicon dioxide (silica) were synthesized. Nanoshells of sample 1 have a diameter of the core of 90 nm and a broad thickness distribution of shells (with an average value of 30 nm), whereas nanoshells of sample 2 have a diameter of the core of 70 nm and a narrow thickness distribution of shells (with an average value of 12 nm). The core diameter, the shell thickness, and the polydispersity of the structure of nanoparticles are estimated by dynamic light scattering. It is shown that the simulation of the optical properties of nanoparticles with their parameters estimated from the dynamic light scattering data makes it possible to obtain good agreement between experimental and theoretical spectra of light scattering. For nanoshells of sample 1, the inhomogeneous broadening of the scattering spectrum is completely determined by the polydispersity; therefore, the bulk constants of gold can be used in simulation of the spectra of such nanoshells. The main mechanism of the broadening for nanoshells of sample 2 is connected with the limitation of the free path length of electrons, whereas the contribution from the thickness distribution of shells can be neglected.