Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142-/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142-/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Formulation of benznidazole-lipid nanocapsules: Drug release, permeability, biocompatibility, and stability studies.

Benznidazole, a poorly soluble in water drug, is the first-line medication for the treatment of Chagas disease, but long treatment periods at high dosages cause several adverse effects with insufficient activity in the chronic phase. According to these facts, there is a serious need for novel benznidazole formulations for improving the chemotherapy of Chagas disease. Thus, this work aimed to incorporate benznidazole into lipid nanocapsules for improving its solubility, dissolution rate in different media, and permeability. Lipid nanocapsules were prepared by the phase inversion technique and were fully characterized. Three formulations were obtained with a diameter of 30, 50, and 100 nm and monomodal size distribution with a low polydispersity index and almost neutral zeta potential. Drug encapsulation efficiency was between 83 and 92 % and the drug loading was between 0.66 and 1.04 %. Loaded formulations were stable under storage for one year at 4 °C. Lipid nanocapsules were found to protect benznidazole in simulated gastric fluid and provide a sustained release platform for the drug in a simulated intestinal fluid containing pancreatic enzymes. The small size and the almost neutral surface charge of these lipid nanocarriers improved their penetration through mucus and such formulations showed a reduced chemical interaction with gastric mucin glycoproteins. LNCs. The incorporation of benznidazole in lipid nanocapsules improved the drug permeability across intestinal epithelium by 10-fold compared with the non-encapsulated drug while the exposure of the cell monolayers to these nanoformulations did not affect the integrity of the epithelium.

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System.

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs' technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro-in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics.

Proteoid biodynamers for safe mRNA transfection via pH-responsive nanorods enabling endosomal escape.

The recent success of mRNA vaccines using lipid-based vectors highlights the importance of strategies for nucleotide delivery under the pandemic situation. Although current mRNA delivery is focused on lipid-based vectors, still they need to be optimized for increasing stability, targeting, and efficiency, and for reducing toxicity. In this regard, other vector systems featuring smart strategies such as pH-responsive degradability and endosomal escape ability hold the potential to overcome the current limitations. Here, we report pH-responsive polymeric nanorods made of amino acid-derivatives connected by dynamic covalent bonds called proteoid-biodynamers, as mRNA vectors. They show excellent biocompatibility due to the biodegradation, and outstanding transfection. The biodynamers of Lys, His, and Arg or monomer mixtures thereof were shown to form nanocomplexes with mRNA. They outperformed conventional transfection agents three times regarding transfection efficacy in three human cell lines, with 82-98% transfection in living cells. Also, we confirmed that the biodynamers disrupted the endosomes up to 10-fold more in number than the conventional vectors. We discuss here their outstanding performance with a thorough analysis of their nanorod structure changes in endosomal microenvironments.

Improving the oral delivery of benznidazole nanoparticles by optimizing the formulation parameters through a design of experiment and optimization strategy.

Chagas disease is a neglected tropical disease affecting the American continent and also some regions of Europe. Benznidazole, approved by FDA, is a drug of choice but its poor aqueous solubility may lead to a low bioavailability and efficacy. Therefore, the aim of this study was to formulate nanoparticles of benznidazole for improving its solubility, dissolution and permeability. A Plackett-Burman design was applied to identify the effect of 5 factors over 4 responses. Then, a Central Composite design was applied to estimate the values of the most important factors leading to the best compromise between highest nanoprecipitation efficiency, drug solubility and lower particle size. The optimized nanoparticles were evaluated for in vitro drug release in biorelevant media, stability studies and transmission electron microscopy. Biocompatibility and permeability of nanoparticles were evaluated on the Caco-2 cell line. The findings of the optimization process indicated that concentration of drug and stabilizer influenced significantly the particle size while concentration of stabilizer and organic/water phase volume ratio mainly influenced the drug solubility. Stability studies suggested that benznidazole nanoparticles were stable after 12 months at different temperatures. Minimal interactions of those nanoparticles and mucin glycoproteins suggested favorable properties to address the intestinal mucus barrier. Cell viability studies confirmed the safety profile of the optimized formulation and showed an increased permeation through the Caco-2 cells. Thus, this study confirmed the suitability of the design of experiment and optimization approach to elucidate critical parameters influencing the quality of benznidazole nanoparticles, which could lead to a more efficient management of Chagas disease by oral route.

Leaky gut model of the human intestinal mucosa for testing siRNA-based nanomedicine targeting JAK1.

Complex in vitro models of human immune cells and intestinal mucosa may have a translation-assisting role in the assessment of anti-inflammatory compounds. Chronic inflammation of the gastrointestinal tract is a hallmark of inflammatory bowel diseases (IBD). In both IBD entities, Crohn's disease and ulcerative colitis, impaired immune cell activation and dysfunctional epithelial barrier are the common pathophysiology. Current therapeutic approaches are targeting single immune modulator molecules to stop disease progression and reduce adverse effects. Such molecular targets can be difficult to assess in experimental animal models of colitis, due to the disease complexity and species differences. Previously, a co-culture model based on human epithelial cells and monocytes arranged in a physiological microenvironment was used to mimic inflamed mucosa for toxicological and permeability studies. The leaky gut model described here, a co-culture of Caco-2, THP-1 and MUTZ-3 cells, was used to mimic IBD-related pathophysiology and for combined investigations of permeability and target engagement of two Janus kinase (JAK) inhibitors, tofacitinib (TOFA) and a JAK1-targeting siRNA nanomedicine. The co-culture just before reaching confluency of the epithelium was used to mimic the compromised intestinal barrier. Delivery efficacy and target engagement against JAK1 was quantified via downstream analysis of STAT1 protein phosphorylation after IFN-γ stimulation. Compared to a tight barrier, the leaky gut model showed 92 ± 5% confluence, a barrier function below 200 Ω*cm2, and enhanced immune response to bacteria-derived lipopolysaccharides. By confocal microscopy we observed an increased accumulation of siJAK1-nanoparticles within the sub-confluent regions leading to uptake into immune cells near the epithelium. A concentration-dependent downregulation of JAK/STAT pathway was observed for siJAK1-nanoparticles (10 ± 12% to 16 ± 12%), whereas TOFA inhibition was 86 ± 2%, compared to untreated cells. By mimicking the status of severely damaged epithelium, like in IBD, the leaky gut model holds promise as a human in vitro system to evaluate the efficacy of anti-inflammatory drugs and nanomedicines.

Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development.

BACKGROUND: The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. RESULTS: In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. CONCLUSION: The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

Drug delivery to the inflamed intestinal mucosa - targeting technologies and human cell culture models for better therapies of IBD.

Current treatment strategies for inflammatory bowel disease (IBD) seek to alleviate the undesirable symptoms of the disorder. Despite the higher specificity of newer generation therapeutics, e.g. monoclonal antibodies, adverse effects still arise from their interference with non-specific systemic immune cascades. To circumvent such undesirable effects, both conventional and newer therapeutic options can benefit from various targeting strategies. Of course, both the development and the assessment of the efficiency of such targeted delivery systems necessitate the use of suitable in vivo and in vitro models representing relevant pathophysiological manifestations of the disorder. Accordingly, the current review seeks to provide a comprehensive discussion of the available preclinical models with emphasis on human in vitro models of IBD, along with their potentials and limitations. This is followed by an elaboration on the advancements in the field of biology- and nanotechnology-based targeted drug delivery systems and the potential rooms for improvement to facilitate their clinical translation.

Surfactant-Free Glibenclamide Nanoparticles: Formulation, Characterization and Evaluation of Interactions with Biological Barriers.

PURPOSE: The aim of this work was to formulate and characterize surfactant-free glibenclamide nanoparticles using Eudragit RLPO and polyethylene glycol as sole stabilizer. METHODS: Glibenclamide nanoparticles were obtained by nanoprecipitation and evaluated in terms of drug content, encapsulation efficiency, apparent saturation solubility, drug release profile, solid state and storage stability. The influence of different stirring speed on the particle size, size distribution and zeta potential of the nanoparticles was investigated. The nanoparticle biocompatibility and permeability were analyzed in vitro on Caco-2 cell line (clone HTB-37) and its interaction with mucin was also investigated. RESULTS: It was found that increasing the molecular weight of polyethylene glycol from 400 to 6000 decreased drug encapsulation, whereas the aqueous solubility and dissolution rate of the drug increased. Particle size of the nanoformulations, with and without polyethylene glycol, were between 140 and 460 nm. Stability studies confirmed that glibenclamide nanoparticles were stable, in terms of particle size, after 120 days at 4°C. In vitro studies indicated minimal interactions of glibenclamide nanoparticles and mucin glycoproteins suggesting favorable properties to address the intestinal mucus barrier. Cell viability studies confirmed the safety profile of these nanoparticles and showed an increased permeation through epithelial cells. CONCLUSION: Taking into consideration these findings, polyethylene glycol is a useful polymer for stabilizing these surfactant-free glibenclamide nanoparticles and represent a promising alternative to improve the treatment of non-insulin dependent diabetes.

Macro- and Microrheological Properties of Mucus Surrogates in Comparison to Native Intestinal and Pulmonary Mucus.

Mucus is a complex hydrogel that acts as a protective barrier in various parts of the human body. Both composition and structural properties play a crucial role in maintaining barrier properties while dictating diffusion of molecules and (nano)materials. In this study, we compare previously described mucus surrogates with the native human airway and pig intestinal mucus. Oscillatory shear rheology was applied to characterize mucus on the bulk macrorheological level, revealing that the artificial airway surrogate deviates from the elastic-dominant behavior of native mucus samples. We circumvented this limitation through the addition of a cross-linking polymer to the surrogate, adjusting the rheological properties closer to those of native mucus. Applying particle tracking microrheology, we further demonstrated that the mechanical properties at the microscale differ significantly between artificial and native mucus. We conclude that proper characterization of mucus and its surrogates is vital for a reliable investigation of nanoparticle-based mucosal drug delivery.

The role of mucus on drug transport and its potential to affect therapeutic outcomes.

A layer of mucus covers the surface of all wet epithelia throughout the human body. Mucus is a hydrogel mainly composed of water, mucins (glycoproteins), DNA, proteins, lipids, and cell debris. This complex composition yields a tenacious viscoelastic hydrogel that lubricates and protects the exposed epithelia from external threats and enzymatic degradation. The natural protective role of mucus is nowadays acknowledged as a major barrier to be overcome in non-invasive drug delivery. The heterogeneity of mucus components offers a wide range of potential chemical interaction sites for macromolecules, while the mesh-like architecture given to mucus by the intermolecular cross-linking of mucin molecules results in a dense network that physically, and in a size-dependent manner, hinders the diffusion of nanoparticles through mucus. Consequently, drug diffusion, epithelial absorption, drug bioavailability, and ultimately therapeutic outcomes of mucosal drug delivery can be attenuated.