Lev A. Dykman

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.

Use of a synthetic foot-and-mouth disease virus peptide conjugated to gold nanoparticles for enhancing immunological response

Foot-and-mouth disease is an acute, highly contagious infection of domestic and wild cloven-hoofed animals, which can be transmitted to humans. In many cases, the existing vaccines are not quite effective. The purpose of this study was to test the possibility of using gold nanoparticles as an antigen carrier and an adjuvant. The immunogenic properties of gold nanoparticles were assessed by conjugating the particles to a synthetic peptide of the VP1 capsid protein of the foot-and-mouth disease virus. The resulting conjugate (with or without the use of complete Freund’s adjuvant), a commercial vaccine, and the native peptide served to immunize guinea pigs. The titer and sensitivity of the raised antibodies were maximal for the combination comprising the nanoparticle–peptide conjugate and complete Freund’s adjuvant. Antibody biosynthesis was accompanied by increased production of proinflammatory cytokines (especially IFN-γ) and by stimulation of the respiratory activity of peritoneal macrophages. The use of gold nanoparticles as a hapten carrier enhanced the immune response even when complete Freund’s adjuvant was not used.

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.

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.

Light Absorption by the Clusters of Colloidal Gold and Silver Particles Formed During Slow and Fast Aggregation

Spectra of absorption (400–800 nm) by the aggregates of colloidal gold (5, 15, and 30 nm in diameter) and silver (20 nm in diameter) particles were studied experimentally and theoretically. It was revealed that, during fast aggregation corresponding to the diffusion-limited cluster aggregation (DLCA), the pattern of spectra is dependent on the size of primary particles. Spectra with the additional absorption maximum in the red region are observed for 15 and 30 nm gold and 20 nm silver particles, while the absorption spectrum for 5 nm particles is characterized by only one maximum shifted to the red region. The slow aggregation resulted in a decrease in plasmon absorption peak with no marked shift to the red region and to the broadening of long-wave absorption wing. From data on electron microscopy, typical branched DLCA-clusters were formed during fast aggregation, whereas small compact aggregates and a noticeable number of single particles were observed in a system during slow aggregation. The computer model of the diffusion-limited cluster-cluster aggregation was used to explain these results. Optical properties of aggregates were calculated using coupled dipole method (CDM or DDA) and the exact method of a multipole expansion. Corrections for the size effect were introduced into the bulk optical constants of metals for nanosized particles. It was shown that a modified version of DDA (Markel et al.,Phys. Rev. B, 1996, vol. 53, no. 5, p. 2425) allows us to explain the pattern of experimental spectra of DLCA-aggregates and their dependence on a monomer size. The exact method was applied to calculate the extinction cross sections of metallic aggregates demonstrating strong electrodynamic interaction between particles. The number of higher multipoles that are required to adequately describe this interaction is much larger than the number of terms of an ordinary Mie series and is the main obstacle to the exact calculation of the spectra of metallic aggregates with a large number of particles.

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.

Multifunctional gold-based nanocomposites for theranostics

Although Au-particle potential in nanobiotechnology has been recognized for the last 15 years, new insights into the unique properties of multifunctional nanostructures have just recently started to emerge. Multifunctional gold-based nanocomposites combine multiple modalities to improve the efficacy of the therapeutic and diagnostic treatment of cancer and other socially significant diseases. This review is focused on multifunctional gold-based theranostic nanocomposites, which can be fabricated by three main routes. The first route is to create composite (or hybrid) nanoparticles, whose components enable diagnostic and therapeutic functions. The second route is based on smart bioconjugation techniques to functionalize gold nanoparticles with a set of different molecules, enabling them to perform targeting, diagnostic, and therapeutic functions in a single treatment procedure. Finally, the third route for multifunctionalized composite nanoparticles is a combination of the first two and involves additional functionalization of hybrid nanoparticles with several molecules possessing different theranostic modalities. This last class of multifunctionalized composites also includes fluorescent atomic clusters with multiple functionalities.