Andrónico Neira-Carrillo

Sulfated polymers in biological mineralization: a plausible source for bio-inspired engineering

Biomineralization leads to the formation of inorganic crystals with unique, ordered, refined shapes that are regulated by specific macromolecules. This process has been a source of inspiration for exploring novel approaches to the fabrication of inorganic-based surfaces and interfaces. Among those macromolecules, sulfated polymers, referred to as proteoglycans, have not received enough attention, although there is increasing evidence of their widespread occurrence in biominerals. Here we examine the available information on the nature, distribution and possible role of sulfated polymers in biomineralization, and highlight new directions to stimulate further research activities.

Antifungal activity of low molecular weight chitosan against clinical isolates of Candida spp.

Chitosan is a natural polymer derived from chitin, a structural component of fungi, insects and shrimp, which exerts antimicrobial effects against bacteria and fungi. The aim of this study was to investigate the in vitro antifungal activity of low molecular weight chitosan (LMWC), and the potential synergy between chitosan and a currently used antifungal drug, fluconazole. The in vitro minimal inhibitory concentrations (MICs) of chitosan and fluconazole against 105 clinical Candida isolates were measured by the broth microdilution method. LMWC exhibited a significant antifungal activity, inhibiting over 89.9% of the clinical isolates examined (68.6% of which was completely inhibited). The species included several fluconazole-resistant strains and less susceptible species such as C. glabrata, which was inhibited at a concentration of 4.8 mg/l LMWC. Although some strains were susceptible at pH 7.0, a greater antifungal activity of LMWC was observed at pH 4.0. There was no evidence of a synergistic effect of the combination of LMWC and fluconazole at pH 7.0. This is the first report in which the antifungal activity of LMWC was investigated with clinical Candida strains. The use of LMWC as an antifungal compound opens new therapeutic perspectives, as the low toxicity of LMWC in humans supports its use in new applications in an environment of pH 4.0-4.5, such as a topical agent for vulvovaginal candidiasis.

Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine.

Peptide vaccines using specific antigens with poor immunogenicity like GnRH-I are unable to develop an effective adaptive immune response and require the presence of adjuvants, essential to lymphocytic activation. Three chitosan formulations were evaluated for their ability as adjuvant of a poor immunogenic peptide vaccine against GnRH-I. Male Sprague-Dawley rats were immunized subcutaneously with recombinant His-GnRH-tandem-repeat peptide in high, low and phosphorylated high molecular weight chitosan solution at 0.5% (w/v). Freunds complete adjuvant was used as a positive control of immune response. Our results suggest that different chitosan formulations as adjuvant, with high or low viscosity degree allow inducing a high and persistent immune response against a poor immunogenic recombinant peptide. We found that the immune response was mediated by a increasing of IgG isotype 1, which were significantly greater than levels presented by the animals immunized with Freunds complete adjuvant. Nevertheless, chitosan with low molecular weight and highest acetylation degree was able to induce an immune response mediated by IgG isotype 2a. Additionally, high molecular weight phosphorylated chitosan, in which the phosphate groups were linked to N-acetyl-d-glucosamine unit, the immune response was reduced. All the immune responses obtained with chitosan as adjuvant were able to neutralize effectively the GnRH hormone proves by reducing of animal steroidogenesis and spermatogenesis demonstrating its capacity to improve immunogenicity in peptide vaccine.

Proteoglycan Occurrence in Gastrolith Of The Crayfish Cherax quadricarinatus (Malacostraca: Decapoda)

ABSTRACT Biomineralized structures are hybrid composites formed and stabilized by the close interaction of the organic and the inorganic phases. Crayfish are good models for studying biomineralization because they develop, in a molting-mineralization cycle, semi-spherical mineralized structures referred to as gastroliths. The organic matrix of these structures consists of proteins, polysaccharides, and lipids. Chitin is the main polysaccharide and is concentrically arranged as fibrous chitin-protein lamellar structures. Although several proteins and low-molecular weight phosphorylated components have been reported to be involved in gastrolith mineralization, the occurrence and role of proteoglycans have not been fully documented. We have immunologically analyzed the proteoglycans in gastrolith matrix extracts and histological cross-sections of the gastrolith, and the forming epithelium during premolt and postmolt stages. The results indicate that gastroliths contain proteoglycans that have dermatan-, chondroitin-4- and 6-, and keratan sulfate glycosaminoglycans. These macromolecules are closely associated with the mineral phase of the gastrolith and are easily removed by decalcification procedures. There is also evidence to indicate that epithelial secretion of some of these molecules is temporally regulated during the molting cycle. However, the precise role of these macromolecules in the calcification and stabilization of the amorphous calcium carbonate phase of the gastrolith remains to be established.

EFFECT OF CROSSLINKED CHITOSAN AS A CONSTRAINED VOLUME ON THE IN VITRO CALCIUM CARBONATE CRYSTALLIZATION

The present work deals with the effect of the constrained volume given by crosslinked chitosan (CHI) as a sphere on the in vitro CaC03 crystallization. Crosslinked CHI was obtained with formaldehyde (FA), glutaraldehyde (GA), epichlorhydrine (EPCH) and poly (propylene glycol) diglycidyl ether (PPDGE) as crosslinking agents. Determination of swelling percentage (%) of the crosslinked CHI spheres was carried out in TRIS buffer at pH 9. Spheres of high molecular weight were prepared using drops of CHI solution on NaOH. In vitro CaC03 crystallization using gas-diffusion method was done. In addition, a synthetically sulphonated containing polymethylsiloxane (S-PMS) was used as modifying additive on the CaC03 crystals growth in a confined space of CHI. The degree of the crosslinking altered the diffusion of C02 gas through the CHI spheres during the CaC03 crystallization resulting in different and specific crystals morphologies

Hybrid biomaterials based on calcium carbonate and polyaniline nanoparticles for application in photothermal therapy

Inorganic materials contain remarkable properties for drug delivery, such as a large surface area and nanoporous structure. Among these materials, CaCO3 microparticles (CMPs) exhibit a high encapsulation efficiency and solubility in acidic media. The extracellular pH of tumor neoplastic tissue is significantly lower than the extracellular pH of normal tissue facilitating the release of drug-encapsulating CMPs in this area. Conducting polyaniline (PANI) absorbs light energy and transforms it into localized heat to produce cell death. This work aimed to generate hybrid CMPs loaded with PANI for photothermal therapy (PTT). The hybrid nanomaterial was synthesized with CaCO3 and carboxymethyl cellulose in a simple, reproducible manner. The CMP-PANI-Cys particles were developed for the first time and represent a novel type of hybrid biomaterial. Resultant nanoparticles were characterized utilizing scanning electron microscopy, dynamic light scattering, zeta potential, UV-vis, FTIR and Raman spectroscopy. In vitro HeLa cells in dark and irradiated conditions showed that CMP-PANI-Cys and PANI-Cys are nontoxic at the assayed concentrations. Hybrid biomaterials displayed high efficiency for potential PTT compared with PANI-Cys. In summary, hierarchical hybrid biomaterials composed of CMPs and PANI-Cys combined with near infrared irradiation represents a useful alternative in PTT.

Evaluation of a multilayered chitosan–hydroxy-apatite porous composite enriched with fibronectin or an in vitro-generated bone-like extracellular matrix on proliferation and diferentiation of osteoblasts

The use of extracellular matrix (ECM) molecules from tissues is an interesting way to induce specific responses of cells grown onto composite scaffolds to promote adhesion, proliferation and differentiation. There have been several studies on the effects on cell proliferation and differentiation of osteoprogenitor cells cultured onto composites, either adding some ECM molecules or grown in the presence of growth factors. Other studies involve the use of osteoblasts cultured on a three‐dimensional (3D) matrix, enriched with ECM molecules produced by the same cells grown previously inside the composite. Here, the effect of enrichment of a novel multilayered chitosan–hydroxyapatite composite with ECM molecules produced by osteoblasts, or the addition of 25 or 50 µg/ml fibronectin to the composite, on proliferation and differentiation of osteoblasts cultured on these composites was studied. The results showed an increase in the number of osteoblasts from day 1 of culture, which was higher in the group grown onto composites enriched with the highest concentration of fibronectin or with ECM molecules produced naturally by osteoblasts cultured previously on them, when compared with the control group. However, this increment tended to decline in all groups after day 7 of culture, the day when they reached the highest peak of proliferation. Differentiation expressed as alkaline phosphatase activity followed the proliferation pattern of the cells cultivated on the scaffolds. The results demonstrate the potential offered by these enriched 3D multilayered composites for improving their ability as bone grafting material. Copyright

CALCIUM CARBONATE GROWTH MODIFICATION BY CONSTITUENTS RELEASES FROM POROUS CELLULOSE FILTER MEMBRANES

Filtration of a suspension of calcium carbonate (CaCO 3 ) or other inorganic solutions are often part of the methodology for recovering crystals during biomimetic mineralization experiments performed by various procedures. However, the use of cellulose filter membranes (FM) may cause a problem in in vitro crystallization experiments, because constituents released from the filters into the filtrates can alter the morphology of CaCO 3 crystals. Therefore, it is possible to misinterpret data obtained when the effect of specific additives tested in the investigation of biomineralization mechanisms. Herein, we present essential information to avoid such misinterpretations of data obtained from mineralization experiments. CaCO 3 was precipitated at room temperature by the gas diffusion method in the presence of FM as support and particularly as filtrates of calcium chloride (CaCl 2 ) obtained from various commercial FM. The occurrence or absence of morphological modifications of the calcite and vaterite crystals obtained with different FM correlates well with the different elemental compositions of the solutions where crystals are grown because of the constituents released from the filters into the filtrate. X-ray photoelectron spectroscopy (XPS) data indicate significant differences in the filter elemental composition. We assume that the observed chronological changes in CaCO 3 crystal modification could be due to incorporation into the calcite lattice of constituents released from the FM, most likely monomers, oligomers, or short-chain polymers.

Calcium carbonate crystallization in tailored constrained environments

Synthesis of inorganic particles using routes inspired by biomineralization is a goal of growing interest. Recently it was demonstrated that the size and geometry of crystallization sites are as important as the structure of charged templating surfaces to obtain particles with controlled features. Most biominerals are formed inside restricted, constrained or confined spaces where at least parts of the boundaries are cell membranes containing phospholipids. In this study, we used a gas diffusion method to determine the effect of different lecithin media on the crystallization of CaCO3 and to evaluate the influence of the spatial arrangement of lecithin molecules on templating CaCO3 crystal formation. By using inorganic synthesis, Raman spectroscopy, dynamic light scattering, electrochemical methods and scanning electron microscopy, we showed that the occurrence of surface-modified calcite crystals and diverse textured vaterite crystals reflects the geometry and spatial distribution of aqueous constrained spaces due to the lecithin assembly controlled by lecithin concentration in an ionized calcium chloride solution under a continuous CO2 diffusion atmosphere. This research shows that by tailoring the assembly of lecithin molecules, as micelles or reversed micelles, it is possible to modulate the texture, polymorphism, size and shape of calcium carbonate crystals.

Control of calcium oxalate morphology through electrocrystallization as an electrochemical approach for preventing pathological disease

AbstractPathological crystallization of calcium oxalate (CaOx) inside the urinary tract is called calculi or kidney stone (Urolithiasis). CaOx exhibits three crystalline types in nature: CaOx monohydrate COM, dihydrate COD and trihydrate COT. COD and COM are often found in urinary calculi, particularly COM. Electrocrystallization has been recently used to perform oriented crystallization of inorganic compounds such as Ca-salts. Although many mineralization methods exist, the mechanisms involved in the control of CaOx polymorphism still remain unclear. Herein, we induced selective electrocrystallization of COD by modifying the electrical current, time and electrochemical cell type. By combining above factors, we established an efficient method without the use of additives for stabilizing non-pathological CaOx crystals. We found notorious stabilization of CaOx polymorphisms with hierarchically complex shape with nano-organization assembly, size and aggregated crystalline particles. Our results demonstrated that, by using an optimized electrochemical approach, this technique could have great potential for studying the nucleation and crystal growth of CaOx through functionalized synthetic polymers, and to develop a novel pathway to evaluate new calculi preventing-compound inhibitors. Graphical abstractElectrocrystallization set-up for modifying the morphology and crystal growth of CaOx particles.