Ethoxy acetalated dextran-based nanocarriers accomplish efficient inhibition of leukotriene formation by a novel FLAP antagonist in human leukocytes and blood

Leukotrienes are pro-inflammatory lipid mediators generated by 5-lipoxygenase aided by the 5-lipoxygenase-activating protein (FLAP). BRP-201, a novel benzimidazole-based FLAP antagonist, inhibits leukotriene biosynthesis in isolated leukocytes. However, like other FLAP antagonists, BRP-201 fails to effectively suppress leukotriene formation in blood, which limits its therapeutic value. Here, we describe the encapsulation of BRP-201 into poly(lactide-co-glycolide) (PLGA) and ethoxy acetalated dextran (Ace-DEX) nanoparticles (NPs), aiming to overcome these detrimental pharmacokinetic limitations and to enhance the bioactivity of BRP-201. NPs loaded with BRP-201 were produced via nanoprecipitation and the physicochemical properties of the NPs were analyzed in-depth using dynamic light scattering (size, dispersity, degradation), electrophoretic light scattering (effective charge), NP tracking analysis (size, dispersity), scanning electron microscopy (size and morphology), UV–VIS spectroscopy (drug loading), an analytical ultracentrifuge (drug release, degradation kinetics), and Raman spectroscopy (chemical attributes). Biological assays were performed to study cytotoxicity, cellular uptake, and efficiency of BRP-201-loaded NPs versus free BRP-201 to suppress leukotriene formation in primary human leukocytes and whole blood. Both PLGA- and Ace-DEX-based NPs were significantly more efficient to inhibit leukotriene formation in neutrophils versus free drug. Whole blood experiments revealed that encapsulation of BRP-201 into Ace-DEX NPs strongly increases its potency, especially upon pro-longed (≥ 5 h) incubations and upon lipopolysaccharide-challenge of blood. Finally, intravenous injection of BRP-201-loaded NPs significantly suppressed leukotriene levels in blood of mice in vivo. These results reveal the feasibility of our pharmacological approach using a novel FLAP antagonist encapsulated into Ace-DEX-based NPs with improved efficiency in blood to suppress leukotriene biosynthesis.

Mice with a humanized immune system are resilient to transplantation of human microbiota

Human gut microbiota has co-evolved with human, and plays important roles in regulating the development and functioning of the host immune system. To study the human-specific microbiome-immunune interaction in an animal model is challenging as the animal model needs to capture both the human-specific immune functions and the human-specific microbiome composition. Here we combined two widely-used humanization procedures to generate a humanized mouse model (HMA-huCD34) with functional human leukocytes developed from engrafted huCD34+ cells and human fecal microbes introduced through fecal microbiota transplantation, and investigated how the two introduced human components interact. We found that the engrafted human leukocytes are resilient to the transplanted human microbes, while reciprocally the transplanted microbial community in the huCD34 mice was significantly different from mice without a humanized immune system. By tracking the colonization of human fecal Bacteroides strains in the mouse gut, we found that the composition of the strain population changes over time, the trajectory of which depends upon the type of mouse. On the other hand, different from Bacteroides, Akkermansia muciniphila exhibited consistent and rapid fixation of a single donor strain in all tested mice, suggesting strong purifying selection common to all mouse types. Our prospect study illustrated the complex interactions between the transplanted microbiome and different host factors, and suggested that the humanized mouse model may not faithfully reproduce the human-specific microbiome-immune interaction.

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.