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.

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5–7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.

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.

Fullerenols are polyhydroxylated derivatives of fullerene (C₆₀(OH)_n) with antioxidant, antiviral, and antibacterial properties and potential biomedical applications due to their solubility and biocompatibility. However, comprehensive assessment of their cytotoxicity is required, particularly regarding their effects on immune system cells. This study investigated the effects of fullerenol C₆₀(OH)₂₄ (MST-Nano, St. Petersburg, Russia) on the viability, apoptosis, and metabolism of THP-1 human monocytic leukemia cells. Cells were treated with concentrations ranging from 0.25 to 1000 µg/mL and incubated for 24, 48, and 72 h. Viability, apoptosis, and nanoparticle association were assessed by flow cytometry; glycolysis and mitochondrial respiration were measured after 24 h on a Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA, USA). Results showed that the effects of fullerenol depend on concentration and exposure time. At 24 h, 750 µg/mL increased viability, while 1000 µg/mL induced apoptosis. After 48 and 72 h, apoptosis increased at concentrations ≥750 µg/mL, with reduced viability. Nanoparticle association correlated with concentration and inversely correlated with viability but was independent of incubation time. Metabolic analysis revealed decreased glycolysis at 750 µg/mL after 24 h, while mitochondrial respiration was unaffected. Thus, our study demonstrated that fullerenol nanoparticles were safe for the THP-1 monocytic cell line up to 500 µg/mL.

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNP) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 Spike-protein in human serum samples by applying a conjugate of CNP with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with Spike-protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing the assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5-7 minutes) of IgG vs Spike-protein with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.

Water-soluble fullerene derivatives such as fullerenol C₆₀(OH)₂₄ are promising candidates for nanomedicine applications, yet their effects on innate immune cells remain poorly characterized. We investigated the interaction of fullerenol with human neutrophils isolated from healthy donors, exposed to concentrations of 0.25–200 μg/mL over 24–72 h. Using multi-parameter flow cytometry, we assessed viability, apoptosis, phagocytic activity, and intracellular reactive oxygen species (ROS) production, complemented by cell-free DPPH radical scavenging assays. Fullerenol was taken up by neutrophils in a concentration- and time-dependent manner. No significant cytotoxicity was observed up to 100 μg/mL, while viability declined at 200 μg/mL. Phagocytosis of opsonized E. coli was preserved at lower concentrations, though a statistically significant negative correlation with fullerenol concentration was detected at higher doses. In cell-free assays, fullerenol scavenged DPPH radicals with an EC₅₀ of 48.90 ± 10.02 μg/mL, exhibiting slower kinetics than Trolox or ascorbic acid. Critically, fullerenol suppressed intracellular ROS production by >33% at 50 μg/mL following PMA stimulation of neutrophils. These findings demonstrate that fullerenol C₆₀(OH)₂₄ combines potent intracellular antioxidant activity with a favorable neutrophil safety profile, supporting its potential application in oxidative stress-related conditions.