The immunostimulatory roles of gold nanoparticles in immunization and vaccination against Brucella abortus antigens.

One effective antigen carrier proposed for use in immunization and vaccination is gold nanoparticles. Prior work has shown that gold nanoparticles themselves have adjuvant properties. Currently, gold nanoparticles are used to design new diagnostic tests and vaccines against viral, bacterial, and parasitic infections. We investigated the use of gold nanoparticles as immunomodulators in immunization and vaccination with an antigen isolated from Brucella abortus. Gold nanoparticles with a diameter of 15 nm were synthesized for immunization of animals and were then conjugated to the isolated antigen. The conjugates were used to immunize white BALB/c mice. As a result, high-titer (1:10240) antibodies were produced. The respiratory and proliferative activities of immune cells were increased, as were the serum interleukin concentrations. The minimum antigen amount detected with the produced antibodies was âˆ¼ 0.5 pg. The mice immunized with gold nanoparticles complexed with the B. abortus antigen were more resistant to B. abortus strain 82 than were the mice immunized through other schemes. This fact indicates that animal immunization with this conjugate enhances the effectiveness of the immune response. The results of this study are expected to be used in further work to examine the protective effect of gold nanoparticles complexed with the B. abortus antigen on immunized animals and to develop test systems for diagnosing brucellosis in the laboratory and in the field.

Immunization of Mice with Gold Nanoparticles Conjugated to Thermostable Cancer Antigens Prevents the Development of Xenografted Tumors.

Gold nanoparticles as part of vaccines greatly increase antigen stability, antigen accumulation in the lymph nodes, and antigen uptake by antigen-presenting cells. The use of such particles as part of anticancer vaccines based on heat shock proteins to increase vaccine effectiveness is timely. We prepared and characterized nanoconjugates based on 15-nm gold nanoparticles and thermostable tumor antigens isolated from MH22a murine hepatoma cells. The whole-cell lysate of MH22a cells contained the main heat shock proteins. BALB/c mice were injected with the conjugates and then received transplants of MH22a cells. The highest titer was produced in mice immunized with the complex of gold nanoparticles + antigen with complete Freund's adjuvant. The immunized mice showed no signs of tumor growth for 24 days. They also showed a decreased production of the INF-γ, IL-6, and IL-1 proinflammatory cytokines compared to the mice immunized through other schemes. This study is the first to show that it is possible in principle to use gold nanoparticles in combination with thermostable tumor antigens for antitumor vaccination. Antitumor vaccines based on thermostable tumor antigens can be largely improved by including gold nanoparticles as additional adjuvants.

Sensor System Based on a Piezoelectric Resonator with a Lateral Electric Field for Virus Diagnostics.

A sensor system based on a piezoelectric resonator with a lateral electric field in the frequency range 6-7 MHz of the electric field for virus detection is described. Through use of the transmissible virus causing gastroenteritis in pigs and specific antibodies, the possibility of detecting the virus in suspension in real time was determined. It was found that the frequency dependence of the real and imaginary parts of the electrical impedance of such a resonator loaded with a virus suspension changes significantly after the addition of specific antibodies to the suspension. No changes are observed if the antibodies are not specific. Thus, the results obtained illustrate the possibility of detecting viruses in situ, directly in the liquid phase, if the change in the real or imaginary parts of the electrical impedance after the addition of antibodies is used as an analytical signal. The possibility of virus detection in the presence of foreign viral particles has been illustrated.

Silica Immobilised Chloro- and Amido-Derivatives of Eremomycine as Chiral Stationary Phases for the Enantioseparation of Amino Acids by Reversed-Phase Liquid Chromatography.

Macrocyclic glycopeptide antibiotics immobilized on silica are one of the effective classes of stationary phases for chiral recognition and HPLC separation of a wide range of optically active compounds. Enantioselectivity primarily depends on the chemical structure of the chiral ligand, immobilization chemistry, and separation conditions. In the present work, three new chiral stationary phases (CSPs) based on macrocyclic antibiotic eremomycin were prepared and investigated for enantioseparation of amino acids. Two eremomycin derivatives, including simple non-substituted amide and bulky adamantyl amide, provided important information on the role of the carboxylic group in the eremomycin structure in the chiral recognition mechanism concerning amino acid optical isomers. One more CSP having a chlorine atom in the same position elucidates the role of the first aromatic ring in the eremomycin structure as a crucial point for chiral recognition. CSP with immobilized chloreremomycin was the most successful among the phases prepared in this work. It was additionally investigated under various separation conditions, including the type and content of the organic solvent in the eluent, the effects of different additives, and the concentration and pH of the buffer. Importantly, an efficient enantioselective separation of amino acids was achieved with pure water as the eluent.

Acoustical Slot Mode Sensor for the Rapid Coronaviruses Detection.

A method for the rapid detection of coronaviruses is presented on the example of the transmissible gastroenteritis virus (TGEV) directly in aqueous solutions with different conductivity. An acoustic sensor based on a slot wave in an acoustic delay line was used for the research. The addition of anti-TGEV antibodies (Abs) diluted in an aqueous solution led to a change in the depth and frequency of resonant peaks on the frequency dependence of the insertion loss of the sensor. The difference in the output parameters of the sensor before and after the biological interaction of the TGE virus in solutions with the specific antibodies allows drawing a conclusion about the presence/absence of the studied viruses in the analyzed solution. The possibility for virus detection in aqueous solutions with the conductivity of 1.9-900 µs/cm, as well as in the presence of the foreign viral particles, has been demonstrated. The analysis time did not exceed 10 min.

Synthesis of Silymarin-Gold Nanoparticle Conjugate and Analysis of its Liver-Protecting Activity.

BACKGROUND: The liver disease problem prompts investigators to search for new methods of liver treatment. INTRODUCTION: Silymarin (Sil) protects the liver by reducing the concentration of free radicals and the extent of damage to the cell membranes. A particularly interesting method to increase the bioavailability of Sil is to use synthesized gold nanoparticles (AuNPs) as reagents. The study considered whether it was possible to use the silymarin-AuNP conjugate as a potential liver-protecting drug. METHODS: AuNPs were conjugated to Sil and the liver-protecting activity of the conjugate was examined. Experimental hepatitis and hepatocyte cytolysis after carbon tetrachloride action were used as a model system, and the experiments were conducted with laboratory animals. RESULTS: For the first time, silymarin was conjugated to colloidal gold nanoparticles (AuNPs). Electron microscopy showed that the resultant preparations were monodisperse and that the mean conjugate diameter was 18-30 nm ± 0.5 nm (mean diameter of the native nanoparticles, 15 ± 0.5 nm). In experimental hepatitis in mice, conjugate administration interfered with glutathione depletion in hepatocytes in response to carbon tetrachloride. It also was conducive to an increase in energy metabolism and stimulated the monocyte-macrophage function of the liver. The results were confirmed by the high respiratory activity of the hepatocytes in cell culture. CONCLUSION: We conclude that the silymarin-AuNP conjugate holds promise as a liver-protecting agent in acute liver disease caused by carbon tetrachloride poisoning.

Synthesis of silymarin-selenium nanoparticle conjugate and examination of its biological activity in vitro.

Silymarin (Sil) was conjugated to selenium nanoparticles (SeNPs) to increase Sil bioavailability. The conjugates were monodisperse; the average diameter of the native SeNPs was ~ 20-50 ± 1.5 nm, whereas that of the conjugates was 30-50 ± 0.5 nm. The use of SeNPs to increase the bioavailability of Sil was examined with the MH-22a, EPNT-5, HeLa, Hep-2, and SPEV-2 cell lines. The EPNT-5 (glioblastoma) cells were the most sensitive to the conjugates compared to the conjugate-free control. The conjugates increased the activity of cellular dehydrogenases and promoted the penetration of Sil into the intracellular space. Possibly, SeNPs play the main part in Sil penetration of cells and Sil penetration is not associated with phagocytosis. Thus, SeNPs are promising for use as a Sil carrier and as protective antigens.

Prospects for the Use of Gold Nanoparticles to Increase the Sensitivity of an Acoustic Sensor in the Detection of Microbial Cells.

The interaction of microbial cells with antibody-gold nanoparticle conjugates in conductive suspensions was experimentally studied by using an acoustic slot-mode sensor. The sensor consisted of a piezoelectric plate with a propagating acoustic wave and a liquid container located above this plate with a given gap. An analysis of the measured parameters of the sensor revealed that the specific interaction of bacterial cells with the conjugates led to a stronger change in the sensor output signal than the specific interaction of bacterial cells with antibodies. The measurements were made for Azospirillum brasilense Sp7 cells in buffer with an initial conductivity of 5-30 µS/cm. The limit of cell detection with the conjugates was 103 cells/mL, and the analysis took about 4 min. The advantage of the sensor is the possibility of repeated use and cleaning of the liquid container without damaging the sensor's elements. These results are promising for use in rapid test systems for the direct detection of microbial cells in actual samples of liquids in medical diagnostics.

Preparation and in vivo evaluation of glyco-gold nanoparticles carrying synthetic mycobacterial hexaarabinofuranoside.

A number of bacterial glycans are specific markers for the detection and the serological identification of microorganisms and are also widely used as antigenic components of vaccines. The use of gold nanoparticles as carriers for glyco-epitopes is becoming an important alternative to the traditional conjugation with proteins and synthetic polymers. In this study, we aimed to prepare and evaluate in vivo glyco-gold nanoparticles (glyco-GNPs) bearing the terminal-branched hexaarabinofuranoside fragment (Ara6) of arabinan domains of lipoarabinomannan and arabinogalactan, which are principal polysaccharides of the cell wall of Mycobacterium tuberculosis, the causative agent of tuberculosis. In particular, we were interested whether the antibodies generated against Ara6-GNPs would recognize the natural saccharides on the cell surface of different mycobacterial strains. Two synthetic Ara6 glycosides with amino-functionalized spacer aglycons differing in length and hydrophilicity were directly conjugated with spherical gold nanoparticles (d = 15 nm) to give two sets of glyco-GNPs, which were used for the immunization of rabbits. Dot assays revealed cross-reactions between the two obtained antisera with the hexaarabinofuranoside and the 2-aminoethyl aglycon used for the preparation of glyco-GNPs. Both antisera contained high titers of antibodies specific for Mycobacteria as shown by enzyme-linked immunosorbent assay using M. bovis and M. smegmatis cells as antigens while there was only a weak response to M. phlei cells and no interaction with E. coli cells. The results obtained suggest that glyco-GNPs are promising agents for the generation of anti-mycobacterial antibodies.

Prospects for the use of spherical gold nanoparticles in immunization.

Recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. We used spherical gold nanoparticles (average diameter, 15 nm) as a platform for the antigen for swine transmissible gastroenteritis virus (TGEV). The literature data demonstrate that immunization of animals with the TGEV antigen coupled to gold nanoparticles (GNPs) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. The contents of γ-IFN, IL-1ß, and IL-6 in animals immunized with GNP-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. The increased concentration of IL-1ß in the immunized animals directly correlated with the activity of macrophages and stimulated B cells, which produce this cytokine when activated. The increased concentration of IL-6 indicates that the injected preparations are stimulatory to cellular immunity. Immunization with the TGEV antigen conjugated to GNPs as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. Furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. The microanatomical changes (increased number of follicles) indicate the activation of the B-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. The degradative processes observed in the animals immunized with TGEV antigen alone are evidence of weak resistance to pathogen attack. These results can be used to develop vaccines against this infection by employing TGEV antigen coupled to gold nanoparticles as a carrier.

Gold nanoparticles as an adjuvant: Influence of size, shape, and technique of combination with CpG on antibody production.

Gold nanoparticles (GNPs) are advantageous as an adjuvant in the design of effective vaccines and in the preparation of high-affinity antibodies to haptens and complete antigens. Another method of activating immunocompetent cells with colloidal gold is to conjugate GNPs with CpG oligodeoxynucleotides (ODNs). We examined how the size and shape of GNPs and various combinations of GNPs and CpG ODNs 1826 affect the immune response. When animals were injected with a model antigen (BSA) coupled to gold nanospheres (diameters, 15 and 50nm), nanorods, nanoshells, and nanostars, the titers of the resultant antibodies differed substantially. The antibody titers decreased in the sequence GNPs-50nm>GNPs-15nm>nanoshells>nanostars>nanorods>native BSA. We conclude that 50 and 15nm gold nanospheres are the optimal antigen carrier and adjuvant for immunization. The highest titer of anti-BSA antibodies was detected in the blood serum of mice immunized simultaneously with BSA-GNP and CpG-GNP conjugates.

Bovine serum albumin adsorbed on eremomycin and grafted on silica as new mixed-binary chiral sorbent for improved enantioseparation of drugs.

A new silica-based, mixed-binary chiral sorbent grafted with the macrocyclic antibiotic eremomycin and bovine serum albumin (BSA) was obtained. The sorbent-filled high-performance liquid chromatography column was capable of enantioseparation of racemic drugs, such as profens, in reversed-phase-chromatography mode. The mixed-binary eremomycin-BSA-sorbent showed better capability for profens enantioseparation as compared with a sorbent containing eremomycin only. BSA grafted onto the sorbent surface significantly reduced retention times of other proteins from the analyte solution, and free proteins (including BSA) injected as analytes were not retained on the column, and subsequently eluted with a dead volume. The drastic difference observed in the binding of profens and other proteins using the sorbent was tested for determination and enantioseparation of profens in artificial-urine solutions.

Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes.

The exposure of Azospirillum brasilense carbohydrate epitopes was investigated by electro-optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO) properties of the suspensions, we used antibodies generated to the complete lipopolysaccharide of A. brasilense type strain Sp7 and also antibodies to the smooth and rough O polysaccharides of Sp7. After 18 hr of culture growth, the EO signal of the suspension treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 hr of culture growth, the strongest EO signal was observed for the cells treated with antibodies to rough O polysaccharide (approximately 46% greater than the control), whereas for the cells treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of the control). These data were confirmed by electron microscopy. The results of the study may have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial biotechnology, when the antigenic composition of the bacterial surface requires close control.

The usage of phage mini-antibodies as a means of detecting ferritin concentration in animal blood serum.

Mini-antibodies that have specific ferritin response have been produced for the first time using sheep's phage libraries (Griffin.1, Medical Research Council, Cambridge, UK). Produced phage antibodies were used for the first time for the development of diagnostic test kits for ferritin detection in the blood of cattle. The immunodot assay with secondary biospecific labeling is suggested as means of ferritin detection in cow blood serum (antiferritin phage antibodies and rabbit antiphage antibodies conjugated with different labels). Сolloidal gold, gold nanoshells, and horse reddish peroxidase used as labels have shown a similar response while detecting concentration of ferritin (0.2 mg/mL). It is shown that the method of solid-phase immunoassay with a visual view of the results allows determination of the minimum concentration of ferritin in the blood of cows at 0.225 g/mL.

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.

Biodynamic parameters of micellar diminazene in sheep erythrocytes and blood plasma.

In this work, we used a preparation of diminazene, which belongs to the group of aromatic diamidines. This compound acts on the causative agents of blood protozoan diseases produced by both flagellated protozoa (Trypanosoma) and members of the class Piroplasmida (Babesia, Theileria, and Cytauxzoon) in various domestic and wild animals, and it is widely used in veterinary medicine. We examined the behavior of water-disperse diminazene (immobilized in Tween 80 micelles) at the cellular and organismal levels. We assessed the interaction of an aqueous and a water-disperse preparation with cells of the reticuloendothelial system. We compared the kinetic parameters of aqueous and water-disperse diminazene in sheep erythrocytes and plasma. The therapeutic properties of these two preparations were also compared. We found that the surface-active substances improved intracellular penetration of the active substance through interaction with the cell membrane. In sheep blood erythrocytes, micellar diminazene accumulated more than its aqueous analog. This form was also more effective therapeutically than the aqueous analog. Our findings demonstrate that use of micellar diminazene allows the injection dose to be reduced by 30%.

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.

Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic glycopeptide antibiotic.

The separation of the enantiomers of several a-amino acids was studied on a new chiral stationary phase (CSP) which is based on the macrocyclic glycopeptide antibiotic eremomycin attached to silica particles. Retention and separation factors were determined under analytical conditions at ambient temperature for different mobile phase compositions. In order to evaluate the potential with respect to preparative separations the adsorption isotherms of D- and L-methionine were determined for one mobile phase composition applying the elution by characteristic point method. The isotherms were validated by comparing experimentally determined elution profiles with predictions based on the equilibrium dispersive model. Finally, the performance of the eremomycin CSP was compared with a commercially available CSP based on the macrocyclic antibiotic teicoplanin. After determining the isotherms of D- and L-methionine also for the teicoplanin phase, the equilibrium dispersive model was used for both CSP to identify optimal operating conditions. For the separation and conditions considered the new eremomycin CSP revealed a better performance compared to the teicoplanin CSP.