Interaction of Graphene Oxide Nanoparticles with Human Mesenchymal Stem Cells Visualized in the Cell-IQ System.

Graphene oxide is a promising nanomaterial with many potential applications. However, before it can be widely used in areas such as drug delivery and medical diagnostics, its influence on various cell populations in the human body must be studied to ensure its safety. We investigated the interaction of graphene oxide (GO) nanoparticles with human mesenchymal stem cells (hMSCs) in the Cell-IQ system, evaluating cell viability, mobility, and growth rate. GO nanoparticles of different sizes coated with linear or branched polyethylene glycol (P or bP, respectively) were used at concentrations of 5 and 25 µg/mL. Designations were the following: P-GOs (Ø 184 ± 73 nm), bP-GOs (Ø 287 ± 52 nm), P-GOb (Ø 569 ± 14 nm), and bP-GOb (Ø 1376 ± 48 nm). After incubating the cells with all types of nanoparticles for 24 h, the internalization of the nanoparticles by the cells was observed. We found that all GO nanoparticles used in this study exerted a cytotoxic effect on hMSCs when used at a high concentration (25 µg/mL), whereas at a low concentration (5 µg/mL) a cytotoxic effect was observed only for bP-GOb particles. We also found that P-GOs particles decreased cell mobility at a concentration of 25 µg/mL, whereas bP-GOb particles increased it. Larger particles (P-GOb and bP-GOb) increased the rate of movement of hMSCs regardless of concentration. There were no statistically significant differences in the growth rate of cells compared with the control group.

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 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.

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.

Measuring the concentration of protein nanoparticles synthesized by desolvation method: Comparison of Bradford assay, BCA assay, hydrolysis/UV spectroscopy and gravimetric analysis.

The 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.

Nuclear magnetic resonance immunoassay of tetanus antibodies based on the displacement of magnetic nanoparticles.

A nuclear magnetic resonance (NMR) immunoassay based on the application of carbon-coated iron nanoparticles conjugated with recognition molecules was designed. The principle of the assay is that ELISA plates are coated with a capture element, and then an analyte is added and detected by conjugating the magnetic nanoparticles with recognition molecules. Afterwards, the elution solution (0.1-M sodium hydroxide) is added to displace the magnetic nanoparticles from the well surfaces into the solution. The detached magnetic nanoparticles reduce transverse relaxation time (T2) values of protons from the surrounding solution. A portable NMR relaxometer is used to measure the T2. Magnetic nanoparticles conjugated with streptavidin, monoclonal antibodies, and protein G were applied for the detection of biotinylated albumin, prostate-specific antigen, and IgG specific to tetanus toxoid (TT). The limit of detection of anti-TT IgG was 0.08-0.12 mIU/mL. The reproducibility of the assay was within the acceptable range (CV < 7.4%). The key novelty of the immunoassay is that the displacement of the nanoparticles from the solid support by the elution solution allows the advantages of the solid phase assay to be combined with the sensitive detection of the T2 changes in a volume of liquid.

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 100-200 nm and 1-5 µm 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.

The effects of human pregnancy-specific ß1-glycoprotein preparation on Th17 polarization of CD4+ cells and their cytokine profile.

BACKGROUND: Pregnancy-specific ß1-glycoproteins are capable of regulating innate and adaptive immunity, exerting predominantly suppressive effects. In this regard, they are of interest in terms of their pharmacological potential for the treatment of autoimmune diseases and post-transplant complications. The effect of these proteins on the main pro-inflammatory subpopulation of T lymphocytes, IL-17-producing helper T cells (Th17), has not been comprehensively studied. Therefore, the effects of the native pregnancy-specific ß1-glycoprotein on the proliferation, Th17 polarization and cytokine profile of human CD4+ cells were assessed. RESULTS: Native human pregnancy-specific ß1-glycoprotein (PSG) at а concentration of 100 µg/mL was shown to decrease the frequency of Th17 (RORγτ+) in CD4+ cell culture and to suppress the proliferation of these cells (RORγτ+Ki-67+), along with the proliferation of other cells (Ki-67+) (n = 11). A PSG concentration of 10 µg/mL showed similar effect, decreasing the frequency of Ki-67+ and RORγτ+Ki67+ cells. Using Luminex xMAP technology, it was shown that PSG decreased IL-4, IL-5, IL-8, IL-12, IL-13, IL-17, MIP-1ß, IL-10, IFN-γ, TNF-α, G-CSF, and GM-CSF concentrations in Th17-polarized CD4+ cell cultures but did not affect IL-2, IL-7, and MCP-1 output. CONCLUSIONS: In the experimental model used, PSG had а mainly suppressive effect on the Th17 polarization and cytokine profile of Th17-polarized CD4+ cell cultures. As Th17 activity and a pro-inflammatory cytokine background are unfavorable during pregnancy, the observed PSG effects may play a fetoprotective role in vivo.

Solid-phase nuclear magnetic resonance immunoassay for the prostate-specific antigen by using protein-coated magnetic nanoparticles.

A solid phase NMR-based sandwich immunoassay for the prostate-specific antigen (PSA) is presented. Carbon-encapsulated iron nanoparticles were functionalized with bovine serum albumin, coupled to monoclonal antibodies, and then used as magnetic labels. A nitrocellulose membrane with 8-µm pores was coated with capture antibodies and subsequently incubated with a serum sample and a suspension of the nanoconjugate. Test strips were placed in a portable homemade NMR relaxometer. Magnetic nanoparticles attached to nitrocellulose decrease the T2 relaxation time of the water protons located inside the pores of the membrane. Thus, T2 is inversely proportional to the concentration of the antigen (PSA) in the sample. The assay can be performed within 4 h. The detection limit is 0.44 ng mL-1. Kallikrein 2, human chorionic gonadotropin, and α-fetoprotein do not interfere. Graphical abstractSchematic representation of NMR relaxometry-based sandwich dot blot immunoassay of a prostate-specific antigen (PSA). Magnetic nanoparticles bound to immunosorbent decrease the transverse relaxation times (T2) of the water protons located within the pores of the membrane. RF coil: radiofrequency coil.

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.

Conjugation of carbon coated-iron nanoparticles with biomolecules for NMR-based assay.

In this work, we developed and optimized conjugates of carbon-coated iron nanoparticles (Fe@C) with streptavidin and monoclonal antibodies. The conjugation procedure included two stages. First, amino groups were grafted onto the carbon shell to facilitate noncovalent sorption of bovine serum albumin (BSA). Further, the covalent attachment of proteins to the BSA layer via glutaraldehyde coupling was performed. It was established and confirmed that the synthesis procedure is reproducible and allows preparation of stable conjugates. The resulting nanoparticles are clusters of Fe@C particles coated by proteins. The size of the clusters is in the range of 100-190 nm and can be controlled via the tuning of conjugation conditions, including pH, BSA-to-Fe@C ratio, etc. Conjugates of Fe@C with streptavidin and monoclonal antibodies (sizes of approximately 140-150 nm) were synthesized. Proton T2 relaxometry was used to detect these conjugates with very high sensitivity due to the magnetic markers, Fe@C. The relaxivity (r2) of different conjugates varied within the range of 290-450 1/s*mM. Conjugate applicability for relaxometry-based assay was confirmed by direct detection of streptococcal protein G and biotinylated BSA in a dot immunoassay.

Highly Stable Conjugates of Carbon Nanoparticles with DNA Aptamers.

Conjugates of carbon nanoparticles and aptamers have great potential in many areas of biomedicine. In order to be implemented in practice, such conjugates should keep their properties throughout long storage period in commonly available conditions. In this work, we prepared conjugates of carbon nanoparticles (CNP) with DNA aptamers using streptavidin-biotin reaction. Obtained conjugates possess superior stability and kept their physical-chemical and functional properties during 30 days at +4 °C and -20 °C. Proposed approach to conjugation allows loading of about 100-120 pM of biotinylated aptamer per 1 mg of streptavidin-coated CNP (CNP-Str). Aptamer-functionalized CNP-Str have zeta potential of -34 mV at pH 7, mean diameter of 168-177 nm, and polydispersity index of 0.080-0.140. High reproducibility of functionalization was confirmed by preparation of several batches of CNP-aptamer with the same size distribution and aptamer loading using independently synthesized parent CNP-Str nanoparticles. Stability of CNP-aptamer conjugates was significantly enhanced by postsynthesis addition of EDTA that prevents nuclease degradation of immobilized aptamers. Obtained nanoparticles were stable at pH ranging from 6 to 10. Optical properties of CNP-aptamer nanoparticles were also studied and their ability to quench fluorescence via Förster resonance energy transfer was shown. Taking into account properties of CNP-aptamer conjugates, we suppose they may be used in both homo- and heterogeneous colorimetric, fluorescent, and aggregation-based assays.

Dot immunoassay for the simultaneous determination of postvaccination immunity against pertussis, diphtheria, and tetanus.

A dot immunoassay for simultaneous semiquantitative detection of IgG against tetanus toxoid (Ttx) and diphtheria toxoid (Dtx) and qualitative detection of anti-Bordetella pertussis IgGs in human blood serum using carbon nanoparticles functionalized with streptococcal protein G was developed. Inactivated B. pertussis cells in suspension form were used as an antigen in the immunoassay. Pertussis, tetanus, and diphtheria antigens were separately spotted onto nitrocellulose strips, and then the immunostrips were successively incubated with blood sera and a suspension of carbon nanoparticles. The immunostrips were then scanned with a flatbed scanner, and the images obtained were processed with ImageJ. One hundred fifty-five venous blood serum samples from children vaccinated with diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccine were tested in comparison with a conventional ELISA and agglutination test. The total time required for analysis of 32 serum samples was less than 3 h. Comparison between the results of the dot immunoassay and the corresponding ELISA/agglutination test revealed a high level of agreement (Cohen's kappa between 0.765 and 0.813). The lower limit of quantification was 0.06 IU/ml for anti-Ttx and anti-Dtx. The intra-assay coefficients of variation were less than 15% for anti-Ttx and anti-Dtx and less than 10% for anti-pertussis. The diagnostic sensitivity of detection of the antibody protection level was 93.5% for anti-Ttx [95% confidence interval (CI) 83.5-97.9%], 92.4% for anti-Dtx (95% CI 80.9297.5%), and 90.2% for anti-pertussis (95% CI 75.9-96.8%). The diagnostic specificity was 90.9% for anti-Ttx (95% CI 57.1-99.5%), 85% for anti-Dtx (95% CI 61.1-96.0%), and 89.3% for anti-pertussis (95%CI 80.8-94.5%). The dot immunoassay developed does not require expensive reading equipment, and allows detection of antibodies against three antigens in a single analysis. The immunostrips can be stored for a long time without changes in the coloration of the spots. Graphical Abstract The assay procedure. BC Bordetella pertussis cell suspension, CNP carbon nanoparticle, Dtx diphtheria toxoid, Ttx tetanus toxoid.

Comparison of anti-pertussis toxin ELISA and agglutination assays to assess immune responses to pertussis.

BACKGROUND: The goal of our study was to compare the following two methods of assessment of pertussis post-vaccination immunity: bacterial agglutination test and pertussis toxin enzyme-linked immunosorbent assay (ELISA). METHODS: The study was carried out in Perm Region, Russia. We measured pertussis immunity using two serological methods: ELISA of IgG to pertussis toxin (PT) and the agglutination test (AT) among 135 children, in the age range from 2 months to 17 years old. The immunization schedule included four doses of DTwP: at 3, 4.5 and 6 months of age and a booster at 18 months. All participants were divided into six age groups. RESULTS: The percentage of samples with IgG level less than the detection limit in vaccinated children was 52.2%. The total seropositivity rate (the percent of children with agglutinin titres ≥1:160) in vaccinated children was 47.8%. Only a weak association was observed between agglutinin and anti-PT IgG titres (R = .3). Neither the primary nor the booster vaccination with DTwP influenced the IgG levels in children. Agglutinin titres significantly increased after vaccination and declined 5 years after the booster dose. Significant growth of IgG concentration was observed in 11-year-olds, indicating the presence of B. pertussis circulation in the childhood population. CONCLUSIONS: Based on the obtained results and the results of other authors, we summarize that anti-PT ELISA should be carefully used to assess the population immunity to pertussis. Currently, there is neither a serological test that accurately determines the protection against pertussis nor a distinctive criterion of protection that can be applied in seroepidemiological studies.