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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.

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Background Pregnancy-specific β1-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 β1-glycoprotein on the proliferation, Th17 polarization and cytokine profile of human CD4+ cells were assessed. Results Native human pregnancy-specific β1-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-1β, 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.

Research paper on sunthesis of protein nanoparticles<div><br><div><b>Abstract</b></div><div>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.<div><br></div></div></div>