Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Preparation and Characterization of Aminoglycoside-Loaded Chitosan/Tripolyphosphate/Alginate Microspheres against E. coli.

Although aminoglycosides are one of the common classes of antibiotics that have been widely used for treating infections caused by pathogenic bacteria, the evolution of bacterial resistance mechanisms and their inherent toxicity have diminished their applicability. Biocompatible carrier systems can help sustain and control the delivery of antibacterial compounds while reducing the chances of antibacterial resistance or accumulation in unwanted tissues. In this study, novel chitosan gel beads were synthesized by a double ionic co-crosslinking mechanism. Tripolyphosphate and alginate, a polysaccharide obtained from marine brown algae, were employed as ionic cross-linkers to prepare the chitosan-based networks of gel beads. The in vitro release of streptomycin and kanamycin A was bimodal; an initial burst release was observed followed by a diffusion mediated sustained release, based on a Fickian diffusion mechanism. Finally, in terms of antibacterial properties, the particles resulted in growth inhibition of Gram-negative (E. coli) bacteria.

Functional Heterogeneity of Mouse and Human Brain OPCs: Relevance for Preclinical Studies in Multiple Sclerosis.

Besides giving rise to oligodendrocytes (the only myelin-forming cell in the Central Nervous System (CNS) in physiological conditions), Oligodendrocyte Precursor Cells (OPCs) are responsible for spontaneous remyelination after a demyelinating lesion. They are present along the mouse and human CNS, both during development and in adulthood, yet how OPC physiological behavior is modified throughout life is not fully understood. The activity of adult human OPCs is still particularly unexplored. Significantly, most of the molecules involved in OPC-mediated remyelination are also involved in their development, a phenomenon that may be clinically relevant. In the present article, we have compared the intrinsic properties of OPCs isolated from the cerebral cortex of neonatal, postnatal and adult mice, as well as those recovered from neurosurgical adult human cerebral cortex tissue. By analyzing intact OPCs for the first time with 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, we show that these cells behave distinctly and that they have different metabolic patterns in function for their stage of maturity. Moreover, their response to Fibroblast Growth Gactor-2 (FGF-2) and anosmin-1 (two molecules that have known effects on OPC biology during development and that are overexpressed in individuals with Multiple Sclerosis (MS)) differs in relation to their developmental stage and in the function of the species. Our data reveal that the behavior of adult human and mouse OPCs differs in a very dynamic way that should be very relevant when testing drugs and for the proper design of effective pharmacological and/or cell therapies for MS.

Unraveling the Structural Landscape of Chitosan-Based Heparan Sulfate Mimics Binding to Growth Factors: Deciphering Structural Determinants for Optimal Activity.

Chitosan sulfates have demonstrated the ability to mimic heparan sulfate (HS) function. In this context, it is crucial to understand how the specific structural properties of HS domains determine their functionalities and biological activities. In this study, several HS-mimicking chitosans have been prepared to mimic the structure of HS domains that have proved to be functionally significant in cell processes. The results presented herein are in concordance with the hypothesis that sulfated chitosan-growth factor (GF) interactions are controlled by a combination of two effects: the electrostatic interactions and the conformational adaptation of the polysaccharide. Thus, we found that highly charged O-sulfated S-CS and S-DCS polysaccharides with a low degree of contraction interacted more strongly with GFs than N-sulfated N-DCS, with a higher degree of contraction and a low charge. Finally, the evidence gathered suggests that N-DCS would be able to bind to an allosteric zone and is likely to enhance GF signaling activity. This is because the bound protein remains able to bind to its cognate receptor, promoting an effect on cell proliferation as has been shown for PC12 cells. However, S-CS and S-DCS would sequester the protein, decreasing the GF signaling activity by depleting the protein or locally blocking its active site.

A holistic approach to unravelling chondroitin sulfation: Correlations between surface charge, structure and binding to growth factors.

Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands.

Ionic Conductivity, Diffusion Coefficients, and Degree of Dissociation in Lithium Electrolytes, Ionic Liquids, and Hydrogel Polyelectrolytes.

The conductive and diffusional behavior of electrolytes in media with different dielectric and viscoelastic properties is investigated. A revised model to separate the contribution of dissociated and nondissociated species to the diffusion coefficients determined with NMR is proposed. Impedance spectroscopy is used to measure the ionic conductivity of lithium salts in aqueous medium, ionic liquids in aprotic solvents, and hydrogel polyelectrolytes. The diffusion coefficients of the species of interest in those systems are determined with multinuclear pulsed-gradient spin-echo (PGSE) NMR. The results are analyzed using the revised model. It is shown that the degree of ionization could be determined directly from measurements of ionic conductivity and diffusion coefficients in very different types of electrolytes and in a wide range of concentrations. Furthermore, these findings support the original Arrhenius hypothesis about electrolytes and show that the assumption of a complete dissociation is not required to describe their conductive behavior. The reduced conductivity observed in hydrogels, at or near swelling equilibrium, compared to that in solutions could be attributed mainly to the hindered ionic mobility caused by the network structure.

Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy.

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds. Graphical abstract Synchrotron IR microspectroscopy can provide insight into the response of neural progenitor cells to synthetic scaffolds.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics.

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.

Ionic Liquid-Based Thermoplastic Solid Electrolytes Processed by Solvent-Free Procedures.

A series of thermoplastic polymer electrolytes have been prepared employing poly(ethylene oxide) (PEO) as a polymer matrix, bis(trifluoromethane sulfonimide) (LiTFSI), and different room-temperature ionic liquids (RTIL) with bis(fluorosulfonyl)imide (FSI) or TFSI anions. This formulation makes them safe and non-flammable. The electrolytes have been processed in the absence of solvents by melt compounding at 120 °C, using sepiolite modified with d-α-tocoferol-polyethyleneglycol 1000 succinate (TPGS-S) as a physical cross-linker of PEO. Several concentrations of RTILs, lithium salt, and TPGS-S have been tested in order to obtain the highest ionic conductivity (σ) without losing electrolytes' mechanical stability. The materials' rheology and ionic conductivity have been extensively characterized. The excellent crosslinking ability of TPGS-S makes the electrolytes behave as thermoplastic materials, even those with the highest liquid concentration. The electrolytes with the highest concentrations of FSI anion present a σ over 10-3 S·cm-1 at 25 °C and close to 10-2 S·cm-1 at 70 °C, and notably behave as solids at temperatures up to 90 °C despite over 65 wt % of their formulation being liquid. The electrolytes thus obtained are safe solid thermoplastics prepared by industrially scalable procedures and are suitable for energy storage devices, proving the adequacy of polymer-based materials as solid electrolytes for batteries or supercapacitors.

Sequential incorporation of metallic cations (Cd2+ and Hg2+) and N-octylamine into titanium phosphate nanoparticles and their subsequent release in acid media.

Titanium phosphate nanoparticles, TPNP, consisting of a NaTi2(PO4)3 core and a shell of hydrogen phosphate and dihydrogen phosphate of titanium, undergo fast hydrolysis in water releasing phosphoric acid. This reaction is inhibited in the presence of metallic ions like Cd2+ or Hg2+, which are able to replace the protons of the shell acid phosphates. The amount of the adsorbed metallic cations could be regulated using counterions of different basicity. The resulting nanoparticles also incorporate NH2(CH2)7CH3 (N-octylamine) at room temperature forming N-octylammonium/phosphate ion pairs, but it was found that at higher cation concentration inside the nanoparticle, a lower amount of amine was adsorbed. The metallic cations and N-octylamine are released in acid media, but the starting material is not fully recovered.

Ion diffusion coefficients model and molar conductivities of ionic salts in aprotic solvents.

In the study of the electric properties of electrolytes, the determination of the diffusion coefficients of the species that intervene in the charge transport process is of great importance, particularly that of the free ions (D(+) and D(-)), the only species that contribute to the conductivity. In this work we propose a model that allows, with reasonable assumptions, determination of D(+) and D(-), and the degree of dissociation of the salt, α, at different concentrations, using the diffusion coefficients experimentally obtained with NMR. Also, it is shown that the NMR data suffice to estimate the conductivity of the electrolytes. The model was checked by means of experimental results of conductivity and NMR diffusion coefficients obtained with solutions of lithium triflate in ethylene and propylene carbonates, as well as with other results taken from the literature.

The Ionic DTI Model (iDTI) of Dynamic Diffusion Tensor Imaging (dDTI).

Measurements of water molecule diffusion along fiber tracts in CNS by diffusion tensor imaging (DTI) provides a static map of neural connections between brain centers, but does not capture the electrical activity along axons for these fiber tracts. Here, a modification of the DTI method is presented to enable the mapping of active fibers. It is termed dynamic diffusion tensor imaging (dDTI) and is based on a hypothesized "anisotropy reduction due to axonal excitation" ("AREX"). The potential changes in water mobility accompanying the movement of ions during the propagation of action potentials along axonal tracts are taken into account. Specifically, the proposed model, termed "ionic DTI model", was formulated as follows. First, based on theoretical calculations, we calculated the molecular water flow accompanying the ionic flow perpendicular to the principal axis of fiber tracts produced by electrical conduction along excited myelinated and non-myelinated axons.Based on the changes in molecular water flow we estimated the signal changes as well as the changes in fractional anisotropy of axonal tracts while performing a functional task.The variation of fractional anisotropy in axonal tracts could allow mapping the active fiber tracts during a functional task. Although technological advances are necessary to enable the robust and routine measurement of this electrical activity-dependent movement of water molecules perpendicular to axons, the proposed model of dDTI defines the vectorial parameters that will need to be measured to bring this much needed technique to fruition.

The effect of antitumor glycosides on glioma cells and tissues as studied by proton HR-MAS NMR spectroscopy.

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning ((1)H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the (1)H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death.

The surface modification of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymers to improve the attachment of urothelial cells.

Biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] substrates were modified to improve the attachment of porcine urothelial cell culture. The pristine copolymer exhibits excellent mechanical properties to replace the bladder tissue, but its surface lacks chemical functionalities to interact with cells. Thus, wet chemical treatments based on NaOH and ethylenediamine in aqueous [ED(aq)] and isopropanol [ED(isoOH)] media to functionalize the P(HB-co-HHx) films surfaces were compared. Among these treatments, short ED(aq) treatment was able to decrease the hydrophobicity, rendering a surface with amino groups and without a significant alteration of the mechanical properties. Furthermore, to enhance the interaction with urothelial cells, laminin derived YIGSR sequence was covalently bound to these amino functionalized substrates. The focal attachment was clearly improved with this last treatment, comparing with those results found with the unmodified and first-step functionalized P(HB-co-HHx).

Influence of the water content on the diffusion coefficients of Li+ and water across naphthalenic based copolyimide cation-exchange membranes.

The transport of lithium ions in cation-exchange membranes based on sulfonated copolyimide membranes is reported. Diffusion coefficients of lithium are estimated as a function of the water content in membranes by using pulsed field gradient (PFG) NMR and electrical conductivity techniques. It is found that the lithium transport slightly decreases with the diminution of water for membranes with water content lying in the range 14 < λ < 26.5, where λ is the number of molecules of water per fixed sulfonate group. For λ < 14, the value of the diffusion coefficient of lithium experiences a sharp decay with the reduction of water in the membranes. The dependence of the diffusion of lithium on the humidity of the membranes calculated from conductivity data using Nernst-Planck type equations follows a trend similar to that observed by NMR. The possible explanation of the fact that the Haven ratio is higher than the unit is discussed. The diffusion of water estimated by (1)H PFG-NMR in membranes neutralized with lithium decreases as λ decreases, but the drop is sharper in the region where the decrease of the diffusion of protons of water also undergoes considerable reduction. The diffusion of lithium ions computed by full molecular dynamics is similar to that estimated by NMR. However, for membranes with medium and low concentration of water, steady state conditions are not reached in the computations and the diffusion coefficients obtained by MD simulation techniques are overestimated. The curves depicting the variation of the diffusion coefficient of water estimated by NMR and full dynamics follow parallel trends, though the values of the diffusion coefficient in the latter case are somewhat higher. The WAXS diffractograms of fully hydrated membranes exhibit the ionomer peak at q = 2.8 nm(-1), the peak being shifted to higher q as the water content of the membranes decreases. The diffractograms present additional peaks at higher q, common to wet and dry membranes, but the peaks are better resolved in the wet membranes. The ionomer peak is not detected in the diffractograms of dry membranes.

Determination of carbon dioxide transport coefficients in liquids and polymers by NMR spectroscopy.

In liquids and in polymeric membranes, a precise determination of their transport properties is of paramount importance. In this work, an NMR method to measure sequentially the solubility and diffusion coefficients of carbon dioxide in liquids (n-alkanes and 1-alkanols) and in polymer membranes (polyethylene, polybutadiene, and polycarbonate) is described. The results show that NMR measurements are very reproducible and in good agreement with those determined by other methods. Considering that the gas permeability is defined as the product of the solubility and diffusion coefficients, the method allows the determination of all transport parameters in an accurate manner. The influence of chain length, viscosity, and solubility parameters on the transport coefficients of [(13)C]O(2) in alkanes and 1-alkanols was also analyzed and compared to those measured in polyethylene.

Surface modification of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer films for promoting interaction with bladder urothelial cells.

Often bladder dysfunction and diseases lead to therapeutic interventions that require partial or complete replacement of damaged tissue. For this reason, the development of biomaterials to repair the bladder by promoting the adhesion and growth of urothelial cells is of interest. With this aim, a modified copolyester of biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] was used as scaffold for porcine urothelial cell culture. In addition to good biocompatibility, the surface of P(HB-co-HV) substrates was modified to provide both, higher hydrophilicity and a better interaction with urothelial cells. Chemical treatments with ethylenediamine (ED) and sodium hydroxide (NaOH) led to substrate surfaces with decreasing hydrophobicity and provided functional groups that enable the grafting of bioactive molecules, such as a laminin derived YIGSR sequence. Physico-chemical properties of modified substrates were studied and compared with those of the pristine P(HB-co-HV). Urothelial cell morphology on treated substrates was studied. The results showed that focal attachment and cell-related properties were improved for peptide grafted polymer compared with both, the unmodified and functionalized copolyester.

Diffusion of small molecules in a chitosan/water gel determined by proton localized NMR spectroscopy.

Proton localized NMR spectroscopy (MRS) has been applied to study the diffusion of three small molecules, caffeine, theophylline and caprolactam, in chitosan gels with different concentration of water. This technique allows the non-destructive monitorization of diffusant concentration as a function of time and location. Concentration profiles were compared with theoretical curves based on solutions of Fick's diffusion equation for the best fitting, with the appropriate boundary conditions. The measured concentration profiles show a good agreement with the Fickian law. Values of the diffusion coefficients D ranging from 6.1×10(-6) to 3.4×10(-6)cm(2)s(-1) depending on chitosan concentration and type of diffusant molecule were determined. In addition, measurements of diffusion coefficients at equilibrium conditions with proton pulsed field gradient NMR methods supported the observed Fickian behavior and showed values of D in excellent agreement with those determined by proton MRS. All these facts demonstrate that proton MRS is an appropriate method for investigating diffusion process in complex systems, such as polymer gels.