Human Chorionic Gonadotropin in the Regulation of T-Helpers Type 17

Chorionic gonadotropin (hCG) is a key pregnancy hormone that regulates steroidogenesis and has immunomodulatory activity. We studied the effects of native and recombinant hCG on the differentiation, proliferation, and production of IL-17 and IFN-ɣ by T-helper cells induced into the phenotype of T-helper type 17 (Th17) in vitro. We found that hCG had no significant effects on the level of Th17 cells, as assessed by RORɣτ expression, and the proliferation of these cells (Ki-67+). In addition, no effects of hCG on the production of IL-17 and IFN-ɣ by T-helpers induced in the Th17 phenotype were found. At the same time, recombinant hCG (100 IU/mL) increased the number of non-Th17 T-helpers (RORγt-Ki-67+). Thus, hCG did not modulate Th17 cells in our experimental model

The Effect of Pristine and Pegylated Graphene Oxide Nanosheets on the Functions of Human Neutrophils

Graphene oxide (GO) is very useful for biomedicine, due to its physicochemical properties; therefore, its interaction with cells of the immune system has beenextensively studied. Many studies have aimed toreduce the undesirable effects of GO through chemical modification, including through polyethylene glycol (PEG) coating. Neutrophils are the first to respond to foreign object invasion in the body. Their main functions are the uptake and destruction of foreign particles, including with the help of reactive oxygen species (ROS).Our study aimed to investigate theengulfment of unmodified graphene oxide (GO) and graphene oxide coated with polyethylene glycol (GO-PEG) by human neutrophils and the effect of nanosheets on the production of ROS.We used sheets of GO (Ossila, Great Britain, average plate size 1-5 μm) and GO-PEG (569 ± 14 nm, PEG coating≈ 20%) at concentrations of 12.5μg/mL, 25μg/mL, and 50 μg/mL. The uptake of nanosheets was assessed by flow cytometry, taking into account the level of background adhesion of nanoparticles. ROS production was evaluated by luminol-dependent chemiluminescence (LCL).It was found that GO (12.5μg/mL, 25μg/mL, and 50 μg/mL) was actively internalized by neutrophils, while the uptake of GO-PEG was not detected. GO and GO-PEG particles (25 μg/mLand 50 μg/mL) reduced the total production of ROS by human leukocytes.Thus, the modifying of GOnanosheets with PEG resulted in the abolishment of their active uptake by neutrophils but did not affect the GO inhibitory effect on their oxidative activity. Keywords: graphene oxide surface modification, pegylated graphene oxide nanosheets, nanoparticle uptake, human neutrophils, of reactive oxygen specie

Human Chorionic Gonadotropin in the Regulation of T-Helpers Type 17

Chorionic gonadotropin (hCG) is a key pregnancy hormone that regulates steroidogenesis and has immunomodulatory activity. We studied the effects of native and recombinant hCG on the differentiation, proliferation, and production of IL-17 and IFN-ɣ by T-helper cells induced into the phenotype of T-helper type 17 (Th17) in vitro. We found that hCG had no significant effects on the level of Th17 cells, as assessed by RORɣτ expression, and the proliferation of these cells (Ki-67+). In addition, no effects of hCG on the production of IL-17 and IFN-ɣ by T-helpers induced in the Th17 phenotype were found. At the same time, recombinant hCG (100 IU/mL) increased the number of non-Th17 T-helpers (RORγt-Ki-67+). Thus, hCG did not modulate Th17 cells in our experimental model

Albumin nanoparticles loaded with hemin as peroxidase mimics for immunoassay

Contemporary immunoassays commonly used in clinical diagnostics mostly utilize enzymes, such as horseradish peroxidase, for signal generation. Numerous research is dedicated to the development of artificial peroxidase-mimicking catalysts with lower cost, high activity, better operational stability, and tunable properties. Herein we synthesized hemin-loaded bovine serum albumin (BSA) nanoparticles and applied them as catalytic labels (nanozymes) in colorimetric immunoassay of anti-tetanus antibodies. Hemin is a key part of the peroxidase catalytic center, possessing peroxidase like-activity. Albumin nanoparticles were loaded with multiple hemin molecules and decorated with Streptococcal protein G. Resulting nanozymes possessed good colloidal stability and allowed for antibody detection in blood serum. The sensitivity of antibody detection was sufficient for the assessment of post-vaccination immunity

Synthesis and Application of Albumin Nanoparticles Loaded with Prussian Blue Nanozymes

Prussian blue nanozymes exhibit peroxidase-like catalytic activity and are therefore considered a stable and inexpensive alternative to natural peroxidases in the enzyme-linked immunosorbent assay (ELISA). In this work, we propose a robust method of Prussian blue nanozyme functionalization, which relies on the entrapment of nanozymes into albumin nanoparticles. The principle of the method is the addition of ethanol to a solution that contains albumin and nanozymes. At a high ethanol concentration solubility of albumin decreases, resulting in the formation of albumin nanoparticles loaded with nanozymes. The hydrodynamic diameter of nanoparticles was between 120 and 230 nm and depended on the nanozyme-to-BSA ratio. Encapsulation efficiency of nanozymes reached 96–99% and up to 190 μg of nanozymes were loaded per 1 mg of nanoparticles. Nanoparticles were stable at pH 5.5–7.5 and upon long-term storage in deionized water. Excellent reproducibility of the synthesis procedure was confirmed by the preparation of three individual batches of Prussian-blue-loaded BSA nanoparticles with almost identical properties. Nanoparticles were functionalized with monoclonal antibodies using glutaraldehyde cross-linking. The resulting conjugates were applied as labels in an ELISA-like assay of tumor marker prostate-specific antigen (PSA). The lower limit of detection was below 1 ng/mL, which enables measurement of PSA in the range of clinically relevant concentrations

Measuring the Concentration of Protein Nanoparticles Synthesized by Desolvation Method: Comparison of Bradford Assay, BCA Assay, hydrolysis/UV Spectroscopy and Gravimetric Analysis

Research paper on sunthesis of protein nanoparticlesAbstractThe desolvation technique is one of the most popular methods for preparing protein nanoparticles for medicine, biotechnology, and food applications. We fabricated 11 batches of BSA nanoparticles and 2 batches of gelatin nanoparticles by desolvation method. BSA nanoparticles from 2 batches were cross-linked by heating at +70 °C for 2 h; other nanoparticles were stabilized by glutaraldehyde. We compared several analytical approaches to measuring their concentration: gravimetric analysis, bicinchoninic acid assay, Bradford assay, and alkaline hydrolysis combined with UV spectroscopy. We revealed that the cross-linking degree and method of cross-linking affect both Bradford and BCA assay. Direct measurement of protein concentration in the suspension of purified nanoparticles by dye-binding assays can lead to significant (up to 50-60%) underestimation of nanoparticle concentration. Quantification of non-desolvated protein (indirect method) is affected by the presence of small nanoparticles in supernatants and can be inaccurate when the yield of desolvation is low. The reaction of cross-linker with protein changes UV absorbance of the latter. Therefore pure protein solution is an inappropriate calibrator when applying UV spectroscopy for the determination of nanoparticle concentration. Our recommendation is to determine the concentration of protein nanoparticles by at least two different methods, including gravimetric analysis.</div

The Effect of PEGylated Graphene Oxide Nanoparticles on the Th17-Polarization of Activated T Helpers

We investigated the direct effect of PEGylated graphene oxide (P-GO) nanoparticles on the differentiation, viability, and cytokine profile of activated T helper type 17 (Th17) in vitro. The subject of the study were cultures of “naive” T-helpers (CD4+) isolated by immunomagnetic separation and polarized into the Th17 phenotype with a TCR activator and cytokines. It was found that P-GO at low concentrations (5 µg/mL) had no effect on the parameters studied. The presence of high concentrations of P-GO in T-helper cultures (25 μg/mL) did not affect the number and viability of these cells. However, the percentage of proliferating T-helpers in these cultures was reduced. GO nanoparticles modified with linear polyethylene glycol (PEG) significantly increased the percentage of Th17/22 cells in cultures of Th17-polarized T helpers and the production of IFN-γ, whereas those modified with branched PEG suppressed the synthesis of IL-17. Thus, a low concentration of PEGylated GO nanoparticles (5 μg/mL), in contrast to a concentration of 25 μg/mL, has no effect on the Th17-polarization of T helpers, allowing their further use for in-depth studies of the functions of T lymphocytes and other immune cells. Overall, we have studied for the first time the direct effect of P-GO nanoparticles on the conversion of T helper cells to the Th17 phenotype

Prussian blue nanozymes with enhanced catalytic activity: size tuning and application in ELISA-like immunoassay

Prussian blue nanozymes possessing peroxidase-like activity gather significant attention as alternatives to natural enzymes in therapy, biosensing, and environmental remediation. Recently, prussian blue nanoparticles with enhanced catalytic activity prepared by reduction of FeCl3/K3[Fe(CN)6] mixture have been reported. These nanoparticles were denoted as ‘artificial peroxidase’ nanozymes. Our study provides insights into the process of synthesis of ‘artificial peroxidase’ nanozymes. We studied how the size of nanozymes and synthesis yield can be controlled via adjustment of the synthesis conditions. Based on these results, we developed a reproducible and scalable method for the preparation of ‘artificial peroxidase’ with tunable sizes allowing the obtaining of nanozymes with enhanced catalytic activity. ‘Artificial peroxidase’ nanozymes modified with gelatin shell and functionalized with affine molecules were applied as labels in colorimetric immunoassays of prostate-specific antigen and tetanus antibodies, enabling detection of these analytes in the range of clinically relevant concentrations. Protein coating provides excellent colloidal stability of nanozymes in physiological conditions and stability upon long-term storage

Modified desolvation method enables simple one-step synthesis of gelatin nanoparticles from different gelatin types with any bloom values

Gelatin nanoparticles found numerous applications in drug delivery, bioimaging, immunotherapy, and vaccine development as well as in biotechnology and food science. Synthesis of gelatin nanoparticles is usually made by a two-step desolvation method, which, despite providing stable and homogeneous nanoparticles, has many limitations, namely complex procedure, low yields, and poor reproducibility of the first desolvation step. Herein, we present a modified one-step desolvation method, which enables the quick, simple, and reproducible synthesis of gelatin nanoparticles. Using the proposed method one can prepare gelatin nanoparticles from any type of gelatin with any bloom number, even with the lowest ones, which remains unattainable for the traditional two-step technique. The method relies on quick one-time addition of poor solvent (preferably isopropyl alcohol) to gelatin solution in the absence of stirring. We applied the modified desolvation method to synthesize nanoparticles from porcine, bovine, and fish with bloom values from 62 to 225 on the hundreds-of-milligram scale. Synthesized nanoparticles had average diameters between 130 and 190 nm and narrow size distribution. Yields of synthesis were 62-82% and can be further increased. Gelatin nanoparticles have good colloidal stability and withstand autoclaving. Moreover, they were non-toxic to human immune cells

Interaction of Graphene Oxide Modified with Linear and Branched PEG with Monocytes Isolated from Human Blood

Multiple graphene-based therapeutics have recently been developed, however potential risks related to the interaction between nanomaterials and immune cells are still poorly understood. Therefore, studying the impact of graphene oxide on various populations of immune cells is of importance. In this work, we aimed to investigate the effects of PEGylated graphene oxide on monocytes isolated from human peripheral blood. Graphene oxide nanoparticles with lateral sizes of 100&ndash;200 nm and 1&ndash;5 &mu;m were modified with linear and branched PEG (GO-PEG). Size, elemental composition, and structure of the resulting nanoparticles were characterized. We confirmed that PEG was successfully attached to the graphene oxide surface. The influence of GO-PEG on the production of reactive oxygen species (ROS), cytokines, phagocytosis, and viability of monocytes was studied. Uptake of GO-PEG by monocytes depends on PEG structure (linear or branched). Branched PEG decreased the number of GO-PEG nanoparticles per monocyte. The viability of monocytes was not altered by co-cultivation with GO-PEG. GO-PEG decreased the phagocytosis of Escherichia coli in a concentration-dependent manner. ROS formation by monocytes was determined by measuring luminol-, lucigenin-, and dichlorodihydrofluorescein-dependent luminescence. GO-PEG decreased luminescent signal probably due to inactivation of ROS, such as hydroxyl and superoxide radicals. Some types of GO-PEG stimulated secretion of IL-10 by monocytes, but this effect did not correlate with their size or PEG structure