Sergio Madrigal-Carballo

Chitosomes loaded with cranberry proanthocyanidins attenuate the bacterial lipopolysaccharide-induced expression of iNOS and COX-2 in raw 264.7 macrophages

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its positively charged amino group. This interaction allows stable covered vesicles (chitosomes) to be developed as a suitable targeted carrier and controlled release system. This study investigated the effect of chitosomes on the activation of cranberry proanthocyanidins (PAC) in Raw 264.7 macrophages. Chitosomes were characterized according to size, zeta potential, PAC-loading, and release properties. Results showed an increase in the net positive charge and size of the liposomes as the concentration of chitosan was increased, suggesting an effective covering of the vesicles by means of electrostatic interactions, as shown by transmission electron microscopy and fluorescence microscopy. About 85% of the PAC that was loaded remained in the chitosomes after release studies for 4 hours in phosphate-buffered saline. Cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are associated with inflammation. Activated RAW 264.7 macrophages increase the expression of COX-2 and iNOS in response to bacterial infection and inflammation; we, therefore, tested the ability of the PAC-loaded chitosomes to attenuate COX-2 and iNOS expression in LPS (lipopolysaccharide)-stimulated macrophages. Increasing the amount of PAC loaded into the chitosomes caused a dose-dependent attenuation of iNOS and COX-2 expression in LPS-stimulated macrophages. A 2% v/v PAC-loaded chitosomes formulation almost completely attenuated the LPS-induced expression of iNOS and COX-2. PAC-loaded chitosomes were more active than PAC alone, suggesting that the macrophage response to LPS occurs after endocytosis of the PAC-loaded chitosomes.

In vitro uptake of lysozyme-loaded liposomes coated with chitosan biopolymer as model immunoadjuvants

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its cationic amino group. This interaction allows developing stable coated vesicles suitable as a targeted carrier and controlled release system for drugs and vaccines. In this work, we studied the effect of chitosan-coated liposomes on the uptake and antigen presentation of hen egg-white lysozyme (HEL) in Peyer’s patches peritoneal macrophages isolated from mice. Chitosan-coated liposomes were characterized according to size, zeta potential, and antigen-loading and release properties. Results showed an increase in the positive net charge and size of the liposomes as the concentration of chitosan was increased, suggesting an electrostatic interaction and an effective coating, followed by fluorescence microscopy. About 85% of the antigen loaded remained in the chitosan-coated liposomes after release studies for 4 hours in phosphate-buffered saline. After 4 hours of preincubation with a T-cell hybridoma line cocultured with murine peritoneal macrophages, only trace amounts of interleukin-2 (IL-2) were detected in the cocultures treated with HEL alone, whereas cocultures treated with HEL-liposomes had an important production of IL-2, and the HEL chitosan-coated liposomes had already reached maximum IL-2 expression. Confocal microscopy studies showed that chitosan-coated liposomes had a higher uptake rate of the fluorescently labeled HEL than uncoated liposomal vesicles after 30 minutes of incubation with the peritoneal macrophages. Since uptake by macrophage cells is the first step in vaccination, our results suggest that the chitosan-coated liposomal system is a potential candidate as an immunoadjuvant for vaccine delivery systems.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages

Abstract In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Cranberry proanthocyanidin-chitosan hybrid nanoparticles as a potential inhibitor of extra-intestinal pathogenic Escherichia coli invasion of gut epithelial cells

Chitosan interacts with proanthocyanidins through hydrogen-bonding, which allows encapsulation and development of stable nanoparticles via ionotropic gelation. Cranberry proanthocyanidins (PAC) are associated with the prevention of urinary tract infections and PAC inhibit invasion of gut epithelial cells by extra-intestinal pathogenic Escherichia coli (ExPEC). We determined the effect of cranberry proanthocyanidin-chitosan hybrid nanoparticles (PAC-CHTNp) on the ExPEC invasion of gut epithelial cells in vitro. PAC-CHTNp were characterized according to size, morphology, and bioactivity. Results showed a decrease in the size of the nanoparticles as the concentration of PAC was increased, indicating that PAC increases cross-linking by hydrogen-bonding on the surface of the chitosan nanoparticles. Nanoparticles were produced with diameters ranging from 367.3 nm to 293.2 nm. Additionally, PAC-CHTNp significantly inhibited the ability of ExPEC to invade the enterocytes by ~80% at 66 μg GAE/mL and by ~92% at 100 μg GAE/mL. Results also indicate that chitosan nanoparticles alone were not significantly different from controls in preventing ExPEC invasion of enterocytes (data not shown) and also there were not significant differences between PAC alone and PAC-CHTNp, suggesting that the new PAC-CHTNp could lead to an increase in the stability of encapsulated PAC, maintain the molecular adhesion of PAC to ExPEC.

Polymer-liposome nanoparticles obtained by the electrostatic bio-adsorption of natural polymers onto soybean lecithin liposomes

This work is focused on the formulation of polymer-liposome nanoparticles based on the electrostatic bio-adsorption of natural polymers onto soybean lecithin liposomes, with potential as novel delivery system for macromolecules, such as proteins. The building up of the polymer-liposome nanoparticles was achieved through the alternating bio-adsorption of natural cationic (chitosan) and neutral (dextran) or anionic (dextran sulphate or alginate) polymer layers on a core composed by anionic nanosized soybean lecithin liposomes. The electrostatic bio-adsorption of natural polymers succeeded in building nanosized, spherical, monodisperse and stable polymer-liposome nanoparticles with cumulative sizes between 357.3 nm ± 25.3 nm and 498.2 nm ± 69.6 nm and surface charges (ζ-potential) between –30.66 mV ± 1.55 mV and –26.74 mV ± 1.04 mV for the liposomal systems composed by alternating layers of chitosan and dextran sulphate or alginate, respectively. Natural-polymer-liposome nanoparticles offer good properties for encapsulation on its liposomal aqueous core and sustained release of a model protein, BSA, in vitro.

Electrospun plant mucilage nanofibers as biocompatible scaffolds for cell proliferation

Electrospun nanofibers (ESNFs) were prepared from mucilage isolated from chan and linaza beans and mozote stem commercially available in Costa Rica. Poly(vinyl alcohol) (PVA) was used as an aiding agent. Mucilage/PVA mixed solutions of different volume ratios (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) were prepared and adjusted to be similar in viscosity and electrical conductivity suitable for electrospinning. Morphology of the ESNFs was examined using scanning electron microscopy (SEM). Fourier transform infrared spectrometer (FTIR) and differential scanning calorimetry (DSC) studies were used to characterize chemical composition and thermal characteristics of the nanofibers (NFs). The ability of the NFs to support fibroblast cell proliferation was investigated in vitro using the optimized mucilage/PVA solutions. Results show plant mucilage-based ESNFs are well-suited for fibroblast cell growth, significantly better than ESNFs of PVA; and the mucilage of chan beans is better than those of mozote and linaza for supporting cell proliferation.