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.

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.

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.

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.