Experimental and in silico approaches for the buffalo whey protein-folic acid complexation elucidation. Molecular changes impacting on protein structure and functionality.

Using a buffalo whey proteins concentrate (BWPC) as a nanocarrier of labile bioactive compounds as vitamins constitutes a very innovative approach with potential application in the food and nutraceutical industries. This work aims to deepen the knowledge of the phenomena occurring in the complexation process of vitamin B9 with BWPC, providing valuable information on the molecular and functional properties of complexes and intervening substances. For such purpose, analytical (SEC-FPLC, Fluorescence spectroscopy, FTIR, DLS, UV-vis spectroscopy) and in-silico methods (molecular docking) were performed to get complementary data. Five types of proteins were identified in the BWPC. Folic acid (FA) interacted with BWPC in buffer pH 7 through H-bonds and hydrophobic interactions, inducing conformational changes and modifying the secondary and tertiary protein structure. The resultant BWPC-FA complexes showed a size distribution in the nanoscale (100-150 nm) with no aggregation. Molecular docking showed that lactoferrin had the highest FA binding affinity. Complexation did not reduce the antioxidant activity of intervening substances. Indeed, the radical scavenging capacity of BWPC-FA was 20 % higher than single BWPC. The obtained results provide relevant data enabling the adding value of the main effluent of buffalo dairy industries.

Molecular interactions involved in the complexation process between buffalo whey proteins concentrate and folic acid.

Using buffalo whey proteins as carrier agents of sensitive molecules raises an interesting approach allowing adding value and minimizing the pollution impact of this by-product. In this context, this work aims to explore the molecular interactions between buffalo whey proteins concentrate (BWPC) and folic acid (FA). For this purpose, fluorescence, UV and FTIR analysis were performed on aqueous or solid dispersions of a buffalo whey protein concentrate (5 µM) (BWPC), with variable concentrations (0-20 µM) of FA. Fluorescence and absorption data were fitted by Stern-Volmer, Beckett, Förster resonance energy transfer, and sphere-of-action models (R2 > 0.9). Derived results suggest that BWPC strongly bind to FA through non-covalent interactions and form ground-state complexes. Additionally, BWPC improves the photostability of FA against UV radiation, and chemical denaturation negatively affects the binding properties. Obtained results encourage further studies of BWPC as carrier agents, which could promote innovative applications for this under-utilized proteins source.

Chitosan-tripolyphosphate nanoparticles designed to encapsulate polyphenolic compounds for biomedical and pharmaceutical applications - A review.

Plant-based polyphenols are natural compounds, present in fruits and vegetables. During recent years, polyphenols have gained special attention due to their nutraceutical and pharmacological activities for the prevention and treatment of human diseases. Nevertheless, their photosensitivity and low bioavailability, rapid metabolism and short biological half-life represent the major limitations for their use, which could be overcome by polyphenols encapsulation (flavonoids and non-flavonoids) into chitosan (CS)-tripolyphosphate (TPP) based nanoparticles (NP). In this review, we particularly focused on the ionic gelation method for the NP design. This contribution exhaustively discusses and compares results of scientific reports published in the last decade referring to ionic gelation applied for the protection, controlled and site-directed delivery of polyphenols. As a consequence, CS-TPP NP would constitute true platforms to transport polyphenols, or a combination of them, to be used for the designing of a new generation of drugs or nutraceuticals.

Biocompatibility analysis of high molecular weight chitosan obtained from Pleoticus muelleri shrimps. Evaluation in prokaryotic and eukaryotic cells.

The search for the exploitation and recycling of biomaterials is increasing for reducing the use of non-renewable resources and minimizing environmental pollution caused by synthetic materials. In this context, Chitosan (CS) being a naturally occurring biopolymer becomes relevant. The aim of the present work was to explore the effects of High Molecular Weight CS (H-CS) from Argentinean shrimp's wastes in prokaryotic and eukaryotic in vitro cell cultures. Ultrastructure of H-CS was analysed by SEM and TEM. In vitro studies were performed in prokaryotic (Lactobacillus casei BL23) and eukaryotic (Caco-2, ARPE-19, EA.hy926 and 3T3-L1) culture cells. High performance microscopic techniques were applied to examine culture cells. No changes in morphology were found in any of the cell types. In addition, fluorescent-dyed H-CS revealed that eukaryotic cells could internalize it optimally. Viability was maintained and proliferation rate even increased for Caco-2, ARPE-19 and 3T3-L1 cells under H-CS treatment. Besides, viability was neither altered in L. casei nor in EA.hy926 cells after H-CS exposure. In conclusion, H-CS could be a suitable biopolymer to be exploited for biomedical or food industry applications.

Resveratrol encapsulation in high molecular weight chitosan-based nanogels for applications in ocular treatments: Impact on human ARPE-19 culture cells.

Oxidative stress and inflammation play a pivotal role in ocular diseases. Resveratrol (RSV) is a natural bioactive that has recently attracted attention due to it potent antioxidant and anti-inflammatory properties. However, RSV presents poor aqueous solubility and chemical instability. Besides, effective drug delivery to the posterior segment of the eye is challenge. Nanotechnology emerges as a possible solution to improve both limitations. Here, we developed and characterized nanogels (NG) based on high molecular weight chitosan (HCS) crosslinked with sodium tripolyphosphate. The distribution size of NG presented a major population around 140 nm with a ζ-potential value of 32 ± 2 mV. TEM and AFM images showed that NG exhibited a rounded morphology. RSV encapsulation efficiency was 59 ± 1%. Photodegradation experiments showed that HCS by its own protects RSV from UV light-induced degradation. Biocompatibility assays revealed that NG were not cytotoxic neither inflammatory in human retinal pigment epithelial cells (ARPE-19), which constitutes the outer blood-retinal barrier. After cellular internalization, we report an endo-lysosomal escape of NG, which is crucial for efficient nanocarriers delivery systems. In conclusion, we envision that HCS based NG could constitute novel carriers for RSV, opening the possibility of its application in ocular diseases.

Biological responses induced by high molecular weight chitosan administrated jointly with Platelet-derived Growth Factors in different mammalian cell lines.

In this work, we studied cellular responses known to be involved in tissue regeneration, such as proliferation, migration and tubulogenesis under High Molecular Weight Chitosan (HMWC) and recombinant Platelet-derived Growth Factor (PDGF) treatments using an in vitro cell culture approach. We also analysed changes in mitochondrial dynamics that could be associated with such biological responses. For this proposes, endothelial human cell lines (EA.hy926 and ECFC) and 3T3-L1 mouse fibroblasts were used. The intracellular uptake of HMWC and their co-localization with acidic compartments were evaluated. Our results show that HMWC enhance PDGF-induced proliferation and cell migration in 3T3-L1 fibroblasts. An increase in PDGF-induced mitochondrial fragmentation was observed in 3T3-L1 cell line, but not in EA.hy926 cells, after the addition of HMWC. Endothelial cells, EA.hy926 and ECFC, potentiate their tubulogenesis capacity with the only addition of HMWC. The HMWC/PDGF-BB treatment notably enhanced tubule formation showing a synergistic effect when act combined in cell culture medium. The knowledge of these cellular responses can be used to design new tissue repair strategies using HMWC and PDGF.

Transcytosis of Bacillus subtilis extracellular vesicles through an in vitro intestinal epithelial cell model.

Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.

Toxic effects of A2E in human ARPE-19 cells were prevented by resveratrol: a potential nutritional bioactive for age-related macular degeneration treatment.

Age-related macular degeneration (AMD) is a late-onset retinal disease and the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial cells (RPE) is a crucial contributing factor responsible for the onset and progression of AMD. The toxic fluorophore N-retinyl-N-retinylidene ethanolamine (A2E), a major lipofuscin component, accumulates in RPE cells with age. Phytochemicals with antioxidant properties may have a potential role in both the prevention and treatment of this age-related ocular disease. Particularly, there is an increased interest in the therapeutic effects of resveratrol (RSV), a naturally occurring polyphenol (3,4',5-trihydroxystilbene). However, the underlying mechanism of the RSV antioxidative effect in ocular diseases has not been well explored. We hypothesized that this bioactive compound may have beneficial effects for AMD. To this end, to investigate the potential profits of RSV against A2E-provoked oxidative damage, we used human RPE cell line (ARPE-19). RSV (25 µM) attenuates the cytotoxicity and the typical morphological characteristics of apoptosis observed in 25 µM A2E-laden cells. RSV pretreatment strengthened cell monolayer integrity through the preservation of the transepithelial electrical resistance and reduced the fluorescein isothiocyanate (FITC)-dextran diffusion rate as well as cytoskeleton architecture. In addition, RSV exhorts protective effects against A2E-induced modifications in the intracellular redox balance. Finally, RSV also prevented A2E-induced mitochondrial network fragmentation. These findings reinforce the idea that RSV represents an attractive bioactive for therapeutic intervention against ocular diseases associated with oxidative stress such as AMD.