A complex of gold nanoparticles with silymarin, a flavonoid-type hepatoprotector isolated from the fruits of milk thistle (Silybum marianum L. Gaertn), was obtained by the method of green synthesis. The resulting conjugate was used to immunize rabbits. After the completion of immunization, the isolated serum immunoglobulins were additionally purified using the method of high performance liquid chromatography. Purified anti-silymarin IgGs were tested for specific antigen binding by dot immunoassay. The detection limit for silymarin was 12.5 ng. The obtained antibodies were labeled with TRITC and used to study the penetration of silymarin into the cells of the reticuloendothelial system by immunofluorescence microscopy. It was demonstrated that silymarin conjugated with gold nanoparticles penetrated into peritoneal macrophages much more efficiently as compared to the native preparation. The results obtained can serve as a basis for creating a new dosage form of silymarin in the form of its conjugate with gold nanoparticles, which enhances hepatoprotective properties due to better penetration of the drug into the cells of the reticuloendothelial system.
Nanotechnology is actively used for diagnosis, treatment and prophylaxis against coronaviruses. To date, methods for producing antibodies and prototypes of vaccines for four types of coronaviruses have been developed. These are: transmissible gastroenteritis coronavirus, avian coronavirus, severe acute respiratory syndrome coronavirus, and SARSCoV-2 coronavirus. Gold nanoparticles can be used as adjuvants to increase the effectiveness of vaccines by stimulating antigen-presenting cells and ensuring controlled antigen release. Thus, gold nanoparticles, which have adjuvant properties, can be an excellent tool in the design of effective vaccines against infectious diseases.
The use of Dunaliella salina microalgae as bioreactors allows to produce gold nanoparticles with anticancer activity by means of green chemistry. The method has been reported for the synthesis of gold nanoparticles using extract of D. salina. This green synthesis technique involves using bright sunlight that we consider as poorly controlled condition. The mechanism of nanoparticles toxicity is not yet elucidated. The toxic effect might arise either from toxic intermediates production during bioreduction of gold ions or from physical adsorption of bioactive molecules on nanoparticles surfaces. This research aimed to optimize gold nanoparticles biosynthesis using aqueous extract of D. salina (AED) and to clarify cytotoxic properties of synthesized nanoparticles. The synthesis of nanoparticles involved heating the reaction mixture and was carried out without using sunlight exposure. The fluorometric alamar blue-based toxicity tests with mammalian cell cultures HeLa and Vero were conducted. The following pollutants were tested: biogenic nanoparticles (AED-AuNPs), 15 nm citrate-capped gold nanoparticles stabilized by aqueous extract of D. salina (Cit-AuNPs@AED), 15 nm citrate-capped gold nanoparticles stabilized by polyethylene glycol (Cit-AuNPs@PEG). We have shown that heating the reaction mixture during bioreduction of HAuCl4 by AED led to the formation of gold nanoparticles with average diameter 12.1±4.1 nm. It has been shown that AED-AuNPs exerted a selective cytotoxic effect on the cancer cell line HeLa. Kidney epithelial cell line Vero appeared to be much more resistant to AED-AuNP than HeLa cells over testconcentration range 50-400 mg Au/L. Cit-AuNPs@AED were found to be non toxic. This evidence indicate that the toxicity of biogenic nanoparticles is likely to be associated with biotransformation of D. salina metabolites during the bioreduction of chloroaurate ions.
CLE peptides are important signaling molecules that regulate the balance of proliferation and differentiation of plant cells. In this work, we conjugated gold nanospheres (15 nm) with a chemically synthesized peptide (12 amino acid residues) that the amino acid sequence corresponds to the natural peptides CLE41/44. The "golden number" for CLE41/44 was 2 Μg/mL. A stable conjugate was used to produce rabbit polyclonal antibody. By the method of dot analysis, the presence of antibodies to CLE41/44 was detected in antiserum. Using Western blot and ELISA methods, the reaction of the obtained antibody with a conjugate of CLE41/44 with BSA molecules was demonstrated. The obtained antibody is can potentially be used to the quantitative determination and identification of the localization of peptides in plant tissues.
Method of speckle-microscopy has been adapted to the problem of detection of Chlamydia trachomatis microbial cells in aqueous suspension and fixed on the glass. Combined system “C. trachomatis bacterial cell”-“monoclonal antibody”- “gold nanoparticle” has been used as a complicated scattering element in the case of formation of biospeckles with a small number of scatterers. Optical model of diffraction of strongly focused Gaussian beam on combined scattering system, containing gold nanoparticles, has been suggested. Simulation of speckles, forming with a small number of scattering nanoparticles, has been carried out. It has been demonstrated that the usage of nanoparticles allows to enhance essentially the output signal of speckle-microscope.
Prototype of laser scanning speckle-microscope has been designed. It has been demonstrated experimentally, that signal of speckle microscope is increased drastically in the presence of gold nanoparticles. It has been demonstrated the unique possibility of detection of a single C. trachomatis cell in a tested sample using speckle-microscopy.
Gold nanoparticles are intensively studied in biomedicine. Assessment of their biocompatibility is highly important. Currently there is lack of evidence, concerning nanotoxicity of ultrasmall gold nanoparticles < 5 nm. Existing data are rather contradictory. The aim of that study was to evaluate the toxicity of 2 nm colloidal gold, using microalga Dunaliella salina. Cellular barriers of that microalga are very similar to animal cells so it might be considered as a valuable model for nanotoxicity testing. Chlorophyll content as a test–function was used. Spectrophotometric method for chlorophyll determination in vivo in suspensions of D.salina cultures was applied. Calculated EC50 48h value of ionic gold was 25.8 ± 0.3 mg Au/L. EC50 value of phosphine-stabilized gold nanoclusters was 32.2 ±1.1 mg Au/L. It was not possible to calculate EC50 for 15 nm citrate gold nanoparticles, as they were non-toxic at all concentrations tested. These results are confirmed by fluorescent –microscopic monitoring of the same probes. It was shown that 10-fold growth of phosphine-stabilized gold nanoparticles (from 2.3 ± 0.9 nm to 21.1 ± 7.5 nm) led to 7-fold decrease of their toxicity.
We describe applications of colloidal gold nanoparticles and silica/gold nanoshells to photothermal therapy and X-ray diagnostics of cancer exemplified by spontaneous tumor of cats and dog. It is shown that the contrast of X-ray images of a cat mammalian tumor can be increased significantly by injection of 3-5-nm gold nanoparticles. We also present preliminary results of a complex approach to treatment a dog mouth cavity melanoma by using immunotherapy together with laser photothermolysis enhanced with silica/gold nanoshells.
We report on the first application of silica-gold nanoshells to a solid-phase dot immunoassay. The assay principle is based
on staining of a drop (1μL ) analyte on a nitrocellulose membrane strip by using silica/gold nanoshells conjugated with
biospecific probing molecules. Experimental example is human IgG (hIgG, target molecules) and protein A (probing
molecules). For usual 15-nm colloidal gold conjugates, the minimal detectable amount of hIgG is about 4ng. By contrast,
for nanoshell conjugates (silica core diameter of 70 nm and gold outer diameter of 100 nm) we have found significant
increase in detection sensitivity and the minimal detectable amount of hIgG is about 0.5 ng.
We report on the time-dependent light scattering from single gold nanospheres (diameter about 90 nm) and nanorods (15x25 nm). Unlike the recent report by S&diaero;nnichsen and Alivisatos (Nano Lett. 5, 301-304, 2005), we observed the dynamic modulation of scattering intensity by using unpolarized dark field microscopy equipped with a digital camera. The changes in light scattering intensity from gold nanorods were caused by 3-dimensional rotational diffusion by contrast to 2-dimensional polarized-scattering modulation observed previously. In the case of 90-nm gold nanospheres, the light scattering modulation was related to the translational diffusion only. By using T-matrix method and Rayleigh approximation, we describe the time-dependent variations in light scattering intensity caused by rotation of nanorods at several scattering and orientation geometry. Finally, we present preliminary data on real-time visualization of active interaction between the living rat peritoneal macrophages and gold nanorods.
A new variant of a technique for in vivo production of antibodies to various antigens with colloidal-gold nanoparticles as carrier is discussed. With this technique we obtained highly specific and relatively high-titre antibodies to different antigens. The antibodies were tested by an immunodot assay with gold nanoparticle markers. Our results provide the first demonstration that immunization of animals with colloidal gold complexed with either haptens or complete antigens gives rise to highly specific antibodies even without the use of complete Freund's adjuvant. These findings may attest to the adjuvanticity of gold nanoparticles itself. We provide also experimental results and discussion aimed at elucidation of possible mechanisms of the discovered colloidal-gold-adjuvanticity effect.
Three procedures for determining the average size of colloidal gold (CG) nanoparticles are described. The first procedure is the dynamic light scattering (DLS) method. The others are based on the estimation of extinction or differential static light scattering (DSLS, at 90 degrees) peak positions. Experimental studies were carried out with four samples of CG (the average DLS-diameters were equal to 15, 20, 25, and 30 nm). In the case of DSLS, the angular slope of the calibration curve "peak position versus particle size" was 2.4 times greater than the extinction curve slope. In experiments with controlled polydispersity, two different colloid mixtures were used. Extinction spectra of samples were almost identical, whereas the DSLS spectra showed noticeable differences with a red shifted maximum. The theoretical simulations by the Mie theory are in agreement with these experimental observations. The DSLS technique seems more reliable and accurate in CG particle sizing, as compared with the usual absorption spectroscopy.
In this work, we report on the optical properties of colloidal gold bioconjugates obtained by attaching protein molecules or oligonucleotide probes to the surface of the particles. Addition of complementary components to the bioconjugate probe results in a specific assembling of conjugates and can be monitored by light scattering and extinction spectra. To this end, we have developed a special attachment to the Specord M-40 spectrophotometer and a corresponding measurement procedure called by us differential light scattering spectroscopy. This method is suitable for the nearly simultaneous recording of extinction and static light scattering spectra of a simple that possesses overlapping spectral bands of absorption and light scattering.
We report on the experimental spectra of light scattered at 90 degrees by colloidal gold particles (diameter 30 nm) and aggregates built from these particles during the salt aggregation process. The time dependence of spectra in the range 350-850 nm was measured by using a luminescence attachment to Specord M-40 spectrophotometer. The aggregation of sol was accompanied by essential increasing the single particle spectrum maximum (near 580 nm), its displacement to the red part in the spectrum, and by a broadening of the long wavelength wing of extinction. To explain these findings, we used a computer diffusion-limited cluster-cluster aggregation model. The optical properties of aggregates including light scattering spectra were computed by the coupled dipole method (CDM or DDA). The bulk optical constants of metals were modified by the size-limiting effect of nanoparticles. Our calculations showed a direct correlation between cluster particle number (Nequals10-50) and intensity of scattered light. However, this correlation failed for 100-particle clusters. It can be supposed that DDA model is not adequate for accurate prediction of light scattering properties of large aggregates.
In this paper, we report on the optical properties of aggregates formed by biospecific interactions like antigen/antibody, with one or both reaction components immobilized on gold particles. In the case of biospecific aggregation, the temporal changes in the absorption spectra differed from those recorded during rapid and slow salt aggregations. As in the case of rapid salt aggregation, the absorption peak decreased and shifted to the red part of the spectrum with simultaneous broadening. However, we did not observe the second red peak of the optical density. According to the transmission electron microscopy data, the slow, rapid, and biospecific aggregations resulted in small clusters with compact structures, branching aggregates of fractal type, and aggregates without direct conductive contacts of primary particles, respectively. It is supposed that the recorded differences in absorption spectra can be explained by the corresponding differences in aggregate structures. We have found a direct correlation between the amount of the second added protein initiating aggregation on the one hand, and the rate of spectral changes on the other. Using these spectral changes, we have plotted a calibration curve for a sufficiently rapid and technically simple quantitative test like sol-particle immunoassay.
The current work was undertaken to develop optical methods for the controlled preparation of gold sols and conjugates with biospecific macromolecules. The extinction spectra of sols with the particle size and axial ratio polydispersity were calculated using Mie's theory, the T-matrix method, and various experimental sets of the bulk gold optical constants modified with regard to size-limiting effects. It was shown that the most generalized model including the size dependence of the imaginary part of the dielectric permeability and the size and shape polydispersity gave good agreement with the experimental extinction spectra for 5-, 10-, 24-, and 40-nm sols, as well as with the size dependence of the position and value of the extinction peak. Electron-microscopic and spectral measurements yielded calibration curves for efficient spectrophotometric control over the particle size and for estimation of the amount of restorer essential for the preparation of particles of a given size. The stabilizing concentrations and the extinction spectra of colloidal gold-ovomucoid conjugates were measured. A simplest two- layered spherical model was employed to elucidate basic changes in sol spectra after conjugation with specific biomacromolecules and to draw some conclusions about the conjugate shell structure.
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