Magnetic nanoclusters coated with albumin, casein, and gelatin: Size tuning, relaxivity, stability, protein corona, and application in nuclear magnetic resonance immunoassay

The surface functionalization of magnetic nanoparticles improves their physicochemical properties and applicability in biomedicine. Natural polymers, including proteins, are prospective coatings capable of increasing the stability, biocompatibility, and transverse relaxivity (r2) of magnetic nanoparticles. In this work, we functionalized the nanoclusters of carbon-coated iron nanoparticles with four proteins: bovine serum albumin, casein, and gelatins A and B, and we conducted a comprehensive comparative study of their properties essential to applications in biosensing. First, we examined the influence of environmental parameters on the size of prepared nanoclusters and synthesized protein-coated nanoclusters with a tunable size. Second, we showed that protein coating does not significantly influence the r2 relaxivity of clustered nanoparticles; however, the uniform distribution of individual nanoparticles inside the protein coating facilitates increased relaxivity. Third, we demonstrated the applicability of the obtained nanoclusters in biosensing by the development of a nuclear-magnetic-resonance-based immunoassay for the quantification of antibodies against tetanus toxoid. Fourth, the protein coronas of nanoclusters were studied using SDS-PAGE and Bradford protein assay. Finally, we compared the colloidal stability at various pH values and ionic strengths and in relevant complex media (i.e., blood serum, plasma, milk, juice, beer, and red wine), as well as the heat stability, resistance to proteolytic digestion, and shelf-life of protein-coated nanoclusters. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Immunoregulatory potential of pregnancy-specific β1-glycoprotein

The embryo, being half an antigenically “foreign” organism, should elicit a maternal immune response. During evolution, however, the mechanisms ensuring successful development of pregnancy have been formed. In particular, among factors providing immune tolerance during pregnancy are some proteins associated with pregnancy. The pregnancy-specific β 1-glycoprotein (PSG, PSG1; SP1; PSβG1) is a dominant fetoplacental protein produced by cyto- and syncytiotrophoblast cells, and it exhibits immunosuppressive properties. Our team of authors possesses a patented method for obtaining native human PSG preparation from blood serum of pregnant women, a mixture of PSG1, PSG3, PSG7, PSG9, and their isoforms and precursors. This review presents an analysis of our results for the period from 2015 to 2020. We studied the immunoregu-latory effects of the obtained PSG preparation at concentrations comparable to those observed in pregnancy (1, 10, 100 |ag/mL). The study was performed with peripheral blood cells obtained from non-pregnant women. It was found that PSG significantly increased the percentage of adaptive Tregs in vitro, as well as expression of CTLA-4, GITR, and production of IL-10 by these cells. It has been shown that PSG has a stimulating effect upon indoleamine-2,3-dioxygenase (IDO) activity of peripheral blood monocytes. For Th17 cells, we have demonstrated that PSG can suppress differentiation and proliferation of these cells, along with reduced production of critical proinflammatory cytokines (IL-8, IL-10, IL-17, IFNγ, MCP-1, TNF α). As for the memory T cells, PSG suppressed CD25 expression and IL-2 production by them, along with simultaneous decreased expression of Gfi1, hnRNPLL genes, thus preventing the formation of the “mature” CD45R0 isoform. PSG has been shown to inhibit naive T cells’ conversion to the terminally differentiated effector subpopulation of helper T cells. When analyzing PSG effects upon cytokine profile of immunocompetent cells, it was found that the protein predominantly suppresses the Th1 cytokine production by the studied cell types, and regulates the Th2 cytokine production in divergent manner. The results obtained are consistent with general concept of immunosuppression during pregnancy. Thus, PSG could be one of the factors preventing formation and implementation of immune response to placental antigens

Effect of pregnancy-specific β1-glycoprotein on myeloid-derived suppressor cell differentiation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that primarily suppress T lymphocytes in healthy pregnancies and pathologies. MDSCs are one of the key regulators of immune responses. Finding ways to control them is important for the treatment of cancer, autoimmune diseases, miscarriage, and post-transplant complications. The mechanisms of immune suppression by MDSC are: expression of CD73, ADAM17, PD -L1, production of Arg 1, iNOS, IDO, IL -10 and TGF-b1.Pregnancy-specific b1-glycoprotein (PSG) has modulatory effects on dendritic cells and macrophages that mediate the shift of T cell phenotypes toward Th2 and Treg. We have previously shown that native PSG suppresses Th17 differentiation and cytokine production, stimulates the production of IDO by monocytes and the differentiation of Tregs.Considering the immunomodulatory properties of PSG and the key role of MDSCs in pathologies, the aim of our work was to investigate the effect of native and recombinant PSG on the differentiation of MDSCs in vitro.MDSCs were differentiated from CD11b+ peripheral blood cells. Cells were cultured for 7 days and received stepwise GM-CSF, IL-1b, and LPS. Native (n) (1; 10 and 100 mg/mL) and recombinant (r) (1 and 10 mg/mL) PSG were introduced into the cultures three days before the end of incubation. Flow cytometry was used to determine the percentage of MDSC among the cells in culture and the percentage of M-, PMN-, and e-MDSC among the total number of MDSCs.It was found that rPSG (1 mg/mL) increased the percentage of MDSCs in culture. Both nPSG (1 and 10 mg/mL) and rPSG (10 mg/mL) increased the proportion of M-MDSC, whereas rPSG (10 mg/mL) decreased the number of PMN-MDSC.Thus, the cytokine background in CD11b+ cell cultures favored the differentiation of predominantly M-MDSC, similar to the tumor microenvironment, whereas native and recombinant PSG enhanced this effect. Thus, nPSG and rPSG are able to modulate the differentiation of MDSCs by increasing their number, mainly due to the monocytic subpopulation. This fact opens perspectives for new research on targeted manipulation of MDSCs

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 gelatin 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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Funding: The reported study was funded by RFBR and Kaliningrad Oblast according to the research project № 19-415-393005 (preparation of fluorescence gelatin nanoparticles and study of their fluorescent properties), by RFBR research project 19-015-00408 (preparation of gelatin nanoparticles by the desolvation method), and by Ministry of Science and Higher Education of the Russian Federation within the framework of the Russian State Assignment under contract No. AAAA-А19-119112290010-7 (assessment of nanoparticles cytotoxicity)

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 their synthesis. 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 and enhanced catalytic activity. 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. © 2022 by the author. Licensee MDPI, Basel, Switzerland.Russian Science Foundation, RSF: 20-75-00029Funding: This study was supported by the Russian Science Foundation, grant 20-75-00029

Study of the graphene oxide nanoparticles effect on luminol-dependent chemiluminescence of human leukocytes

Graphene and its derivatives are increasingly used in biomedical research. Therefore, the mechanisms and consequences of the interaction of graphene nanoparticles with living objects are intensively studied. The immune system is involved in protecting the body and regulating its functions, so the question of the effect of graphene and its derivatives on immune cells is crucial. The specific response of monocytes, macrophages, and neutrophils to a stimulus is to increase the production of reactive oxygen species (ROS). Published data on graphene oxide (GO) and polyethylene glycol-modified graphene oxide (GO-PEG) effects on peripheral blood leukocytes are scarce and contradictory. It is due to variations in objects and conditions of study, along with the difference in particle concentrations. Thus, it was essential to evaluate the GO and GO-PEG effect on ROS production by human leukocytes. Our study aimed at the effect of particles of unmodified and PEG-modified graphene oxide (GO and GO-PEG) on the ROS production by peripheral blood leukocytes in not-stimulated and stimulated luminoldependent chemiluminescence (LCL) tests. ROS production was stimulated by opsonized zymosan (OZ). A hydrogen peroxide-luminol system was used for assessing the independent effect of GO nanoparticles on the quenching of ROS luminescence. Pristine GO (Ossila, Great Britain) nanoparticles were PEG-modified (GO-PEG). The average size of the GO flakes was 1-5 µm, the GO-PEG-flakes 569±14 nm, and the amount of PEG covering was ~ 20%. Nanoparticles were used at concentrations of 5; 2.5; 1.25 µg/ml. It has been established that GO-PEG nanoparticles in concentrations of 2.5 and 5 µg/ml suppressed ROS production in the spontaneous LCL test. At the same time, the GO effects showed a visible but a not significant tendency to inhibition of LCL. Similar results were obtained in the stimulated LCL test. However, when analyzing the process kinetics, both GO-PEG and GO decreased the ROS production, but mainly in the first minutes of the test. When analyzing the quenching effect of the LCL reaction in a cell-free system, there was no significant effect of GO and GO-PEG nanoparticles. Thus, the general vector of the obtained effects was associated with the suppression of ROS production. GO-PEG ROS-decreasing effects were more pronounced in comparison with unmodified GO. In general, we have confirmed the antioxidant effects of GO and GO-PEG using the LCL method. We can assume that in addition to the actual antioxidant effect of graphene nanoparticles, ROS production decreases due to the rapid GO uptake and blocking of several intracellular signals that induce an oxidative burst

Effect of short PSG peptide fragments on the cytokine profile in Wistar rats during allogeneic transplantation <i>in vivo</i>

Pregnancy-specific beta-1-glycoprotein (PSG) is a protein with pleiotropic biological effects, particularly immunoregulatory and immunosuppressive potential. The use of recombinant PSG may exert therapeutic effects in experimental animals with induced autoimmune diseases. Recently, a search for the biological effects of short linear motifs (SLiMs) has become a new strategy for designing the pharmacological compounds. Tetrapeptide regions have been identified in the primary structure of several PSGs: YQCE, YECE and YACS, these SLiMs exhibit immunomodulatory activity. The aim of our study was to evaluate the prospectives for usage of PSG peptide fragments as pharmacological agents to modulate transplant immunity. We used an original model of host-versus-graft response in male Wistar rats transplanted with bone marrow, without prior conditioning treatment of recipients. We used a cocktail of the PSG peptide fragments administered to Wistar rats in the course of allogeneic bone marrow transplantation (BM) in dynamic manner, evaluating the cytokine profile as an integral index of immune response. Cytokine levels were determined by multiplex method using Bio-Plex ProTM Rat 23-Plex kit. Statistical processing of the data was performed by means of two-way analysis of variance and Tukey’s post hoc test for multiple comparisons. We have found that the levels of pro-inflammatory cytokines (IFNγ, IL-1α, IL-1β, IL-18), as well as the contents of G-CSF, GM-CSF and IL-7 were increased in the animals injected with BM only. In the group of animals injected with BM + PSG peptides, an increase in IFNγ, IL-6, TNFα was observed, which decreased by the end of the experiment. Increased levels of antiinflammatory cytokines IL-4 and IL-13 were detected in blood serum of the animals on day +14. Moreover, administration of PSG peptides also led to increase in IL-2, M-CSF, MCP-1, and RANTES levels on day 14 from the beginning of the experiment, and to a gradual decrease in their levels till the end of the experiment. Meanwhile, control group showed a marked tendency for increase of these and other cytokines. Thus, it was shown that the use of PSG peptides upon development of immune response to BM allograft may promote a return to normal levels for the most cytokines studied, thus presuming the immunopharmacological potential of these peptides. The obtained data can be used to develop a pharmacological preparation of the studied peptides to correct the imbalance of immune system

ROLE OF GLYCODELIN IN REGULATION OF MYELOIDDERIVED SUPPRESSOR CELL DIFFERENTIATION

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&amp;D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation

THE ROLE OF PREGNANCY-SPECIFIC GLYCOPROTEIN IN REGULATION OF MOLECULAR GENETIC DIFFERENTIATION MECHANISMS OF IMMUNE MEMORY T CELLS

The role of pregnancy-specific β1-glycoprotein (PSG) in the regulation of molecular genetic factors determining the functional activity of naїve T cells and T cells of immune memory in vitro was studied. Human PSG was isolated with a proprietary immuno-purification method using a biospecific sorbent followed by removing of immunoglobulin contamination with a HiTrapTM Protein G HP column. Physiological concentrations of PSG were used in the experiments. They corresponded to PSG levels in the peripheral blood of pregnant woman: 1, 10 and 100 μg/ml (I, II, III trimester, respectively). The objects of study were monocultures of naїve T cells (CD45RA+) and memory T cells (CD45R0+), obtained by immunomagnetic separation from the peripheral blood of women of reproductive age.It was established that at the level of naїve T cells (CD45RA+) PSG inhibited the expression of CD28 (1, 10, 100 μg/ml) and CD25 (100 μg/ml), without affecting the interleukin-2 (IL-2) production by these cells. At the same time, PSG in all concentrations studied suppressed the expression of CD25 at the immune memory T-cell (CD45R0+) surface but increased the IL-2 production. Expression of U2af1l4, Gfi1, hnRNPLL genes regulating the alternative splicing of the Ptprc gene encoding CD45 was also evaluated. It was found, that PSG reduced the expression of the Gfi1 (1, 10, 100 μg/ml), hnRNPLL (10, 100 μg/ml) genes, but increased the expression of the U2af1l4 gene (1, 10, 100 μg/ml) in the naїve T cells. It was shown that at the immune memory T-cells’ level the effects were similar, with PSG rendering them in all concentrations used. The revealed changes in the mRNA transcription of U2af1l4, Gfi1 and hnRNPLL genes in the studied T cell subsets may lead to the inhibition of CD45 “mature” isoform formation – CD45R0.Thus, PSG reduces the functional activity of naїve T cells and immune memory T cells associated with the expression of costimulation/activation molecules CD25 and CD28 and is involved in the regulation of Ptprc gene alternative splicing, which determines the ratio of CD45 molecule variants. Apparently, using these mechanisms, PSG regulates the functional activity of the memory T cell circulating pool, which is potentially capable of carrying out antigen-specific cytotoxic reactions against fetal antigens in vivo. In general, the data obtained broadens the notion of the PSG role in the regulation of molecular-genetic mechanisms of naїve T cells and immune memory T cells differentiation

GRAPHENE OXIDE NANOPARTICLES IN THE REGULATION OF THE OXIDATIVE ACTIVITY OF HUMAN MONOCYTES

Graphene-based materials have an opportunity for use in biomedicine, thanks to their properties. Nevertheless, due to its cytotoxic effects, the use of graphene-based drugs is problematic. However, the surface modification of graphene oxide (GO) nanoparticles with a polyethyleneglycol (PEG) is one way to reduce the harmful effects of graphene on cells. Applying nanoparticles implies their interaction with the immune system, which protects the body. Monocytes are innate immunity cells and the first line of defenсe of the human organism from microorganisms and other alien objects. One of the monocytes’ reactions to a stimulus of any nature is to produce reactive oxygen species (ROS). Published data shows an incomplete picture of modified graphene oxide nanoparticles’ effects on ROS formation by human monocytes. Thus, it was essential to evaluate the pegylated graphene oxide (GO-PEG and GO-8armedPEG) effect on ROS production by human monocytes, assessed by the luminol-dependent chemiluminescence (LCL). The objects of the study were CD14+-cells isolated from mononuclear cells of healthy donors. ROS production was stimulated by opsonized zymosan (OZ), spontaneous LCL was used as a control. PEG-modified (GO-PEG and GO-8armedPEG) GO nanoparticles with sizes of 100-200 nm (“small”) and 1-5 μm (“big”) with PEG covering ~ 20% were used at concentrations of 5 and 25 μg/ml. The study showed that small size nanoparticles at a low concentration of 5 μg/ml and big nanoparticles coated with 8-armed PEG at both concentrations have a significant suppressive effect on spontaneous ROS production. In the stimulated LCL reaction variant, it was found that small nanoparticles (25 μg/ml) also have a suppressive effect on ROS production, such as big-sized particles coated with linear PEG at the same concentration. Thus, we have established for the first time that graphene oxide nanoparticles functionalized with PEG are capable of inhibiting the ROS production by human monocytes, and therefore, we can speak of the antioxidant activity of GO-PEG