Cryoprotective Polysaccharides with Ordered Gel Structures Induce Ice Growth Anticipation and Survival Enhancement during Cell Cryopreservation.

This work cross-correlated rheological, thermodynamic, and conformational features of several natural polysaccharides to their cryoprotective performance. The basis of cryoprotection of FucoPol, pectin, and agar revealed a causal combination of (i) an emerging sol-gel transition (p = 0.014) at near-hypothermia (4 °C), (ii) noncolligative attenuated supercooling of the kinetic freezing point of water (p = 0.026) supporting ice growth anticipation, and (iii) increased conformational order (p < 0.0001), where helix-/sheet-like features boost cryoprotection. FucoPol, of highest cryoprotective performance, revealed a predominantly helical structure (α/ß = 1.5) capable of forming a gel state at 4 °C and the highest degree of supercooling attenuation (TH = 6.2 °C). Ice growth anticipation with gel-like polysaccharides suggests that the gel matrix neutralizes elastic deformations and lethal cell volumetric fluctuations during freezing, thus preventing the loss of homeostasis and increasing post-thaw viability. Ultimately, structured gels capable of attenuated supercooling enable cryoprotective action at the polymer-cell interface, in addition to polymer-ice interactions. This rationale potentiates implementing alternative, biobased, noncytotoxic polymers in cryobiology.

Elevated fucose content enhances the cryoprotective performance of anionic polysaccharides.

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection.

Ice modulatory effect of the polysaccharide FucoPol in directional freezing.

Directional freezing harnesses crystal growth development to create aligned solid structures or etchable patterns, useful for directed ice growth in cryobiology and cryoprinting for tissue engineering. We have delved into the ice-modulating properties of FucoPol, a fucose-rich, bio-based polysaccharide. Previous research on FucoPol revealed its non-colligative hysteresis in kinetic freezing point, reduced crystal dimensions and cryoprotective effect. Here, FucoPol reshaped developing sharp, anisotropic obloid ice dendrites into linearly-aligned, thin, isotropic spicules or tubules (cooling rate-dependent morphology). The effect was enhanced by increased concentration and decreased cooling rate, but major reshaping was observed with 5 µM and below. These structures boasted remarkable enhancements: uniform alignment (3-fold), tip symmetry (5.9-fold) and reduced thickness (5.3-fold). The ice-modulating capability of FucoPol resembles the Gibbs-Thomson effect of antifreeze proteins, in particular the ice reshaping profiles of type I antifreeze proteins and rattlesnake venom lectins, evidenced by a 52.6 ± 2.2° contact angle (θ) and spicular structure generation. The high viscosity of FucoPol solutions, notably higher than that of sucrose, plays a crucial role. This viscosity dynamically intensifies during directional freezing, leading to a diffusion-limited impediment that influences dendritic formation. Essentially, the ice-modulating prowess of FucoPol not only reinforces its established cryoprotective qualities but also hints at its potential utility in applications that harness advantageous ice growth for intentional structuring. For instance, its potential in cryobioprinting is noteworthy, offering an economical, biodegradable resource, of easy removal, sidestepping the need for toxic reagents. Moreover, FucoPol fine-tunes resulting ice structures, enabling the ice-etching of biologically relevant patterns within biocompatible matrices for advanced tissue engineering endeavors.

Cosmetic emulsion based on the fucose-rich polysaccharide FucoPol: Bioactive properties and sensorial evaluation.

In this study, the physicochemical characteristics, bioactive properties, and sensorial evaluation of a O/W cosmetic formulation containing FucoPol, a fucose-containing bacterial polysaccharide, were assessed. The stability of the FucoPol-based cream, named F-cream, was demonstrated over a period of 2 months at different temperatures (4, 20 and 30 °C), during which it maintained the organoleptic characteristics and pH (5.88-6.19), with minimal variations on the apparent viscosity. Furthermore, no breaking mechanisms occurred upon centrifuging the samples (accelerated stability test) kept at 4 °C and at 30 °C for 60 days. The F-cream presented a shear-thinning and solid-liquid behavior consistent with its envisaged use for topical applications, proving to be a suitable candidate for an anti-aging application due to its antioxidant capacity and effective photoprotection, maintaining cellular preservation. Moreover, the formulation was proven non-cytotoxic for HaCaT cells at concentrations between 0.78 and 12.5 mg/mL, promoting HFFF2 cell migration (46-70 % of wound closure) at a concentration of 2.5 mg/mL, and HaCaT cell migration at a concentration of 10 mg/mL (95-98 % of wound closure). Upon application over the skin, the F-cream provided a hydration and softness with desired spreadability with no residues after application. These findings show that FucoPol has good potential to be used as a functional and/or active ingredient in cosmetic formulations, forming an emulsified cream with appealing sensorial properties that can act as a moisturizer with photoprotection, antioxidant, and regeneration properties.

Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant.

Metastable supercooling has emerged as a transformative technique for ice-free biopreservation, but issues of stability inherent to the stochastic nature of ice formation have thus far limited its translation out of the laboratory. In this work, we explore the influence of the bio-based carbohydrate polymer FucoPol on aqueous supercooling using an isochoric nucleation detection technique. We show that FucoPol, a high-molecular-weight, fucose-rich polysaccharide, which has previously been shown to reduce average ice crystal sizes after nucleation, also induces a concentration-dependent stabilization of metastable supercooled water, as evidenced by both a significant reduction in nucleation stochasticity (i.e., the spread in temperatures over which the system will nucleate upon cooling) and a corresponding increase in the predicted induction time of nucleation. FucoPol is found to confine the stochasticity of ice nucleation to a narrow, well-defined band of temperatures roughly one-third as wide as that of pure water under identical conditions. Importantly, this substantial reduction in stochasticity is accompanied by only a minimal (<1 °C) change in the average nucleation temperature, suggesting that this effect is distinct from colligative freezing point depression. Reducing and characterizing the stochasticity of aqueous supercooling is essential to the engineering design of practical biopreservation protocols, and the results reported herein suggest that high-viscosity polymer systems may provide a powerful and largely unexplored lever by which to manipulate metastable-equilibrium phase change kinetics at subzero temperatures.

Antioxidant Potential of the Bio-Based Fucose-Rich Polysaccharide FucoPol Supports Its Use in Oxidative Stress-Inducing Systems.

Reactive oxygen species (ROS) are dangerous sources of macromolecular damage. While most derive from mitochondrial oxidative phosphorylation, their production can be triggered by exogenous stresses, surpassing the extinction capacity of intrinsic antioxidant defense systems of cells. Here, we report the antioxidant activity of FucoPol, a fucose-rich polyanionic polysaccharide produced by Enterobacter A47, containing ca. 17 wt% of negatively charged residues in its structure. Ferric reducing antioxidant power (FRAP) assays coupled to Hill binding kinetics fitting have shown FucoPol can neutralize ferricyanide and Fe3+-TPTZ species at an EC50 of 896 and 602 µg/mL, respectively, with positive binding cooperativity (2.52 ≤ H ≤ 4.85). This reducing power is greater than most polysaccharides reported. Moreover, an optimal 0.25% w/v FucoPol concentration shown previously to be cryo- and photoprotective was also demonstrated to protect Vero cells against H2O2-induced acute exposure not only by attenuating metabolic viability decay, but also by accentuating post-stress proliferation capacity, whilst preserving cell morphology. These results on antioxidant activity provide evidence for the biopolymer's ability to prevent positive feedback cascades of the radical-producing Fenton reaction. Ultimately, FucoPol provides a biotechnological alternative for implementation in cryopreservation, food supplementation, and photoprotective sunscreen formula design, as all fields benefit from an antioxidant functionality.

On the Development of Selective Chelators for Cadmium: Synthesis, Structure and Chelating Properties of 3-((5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)amino)benzo[d]isothiazole 1,1-dioxide, a Novel Thiadiazolyl Saccharinate.

Aquatic contamination by heavy metals is a major concern for the serious negative consequences it has for plants, animals, and humans. Among the most toxic metals, Cd(II) stands out since selective and truly efficient methodologies for its removal are not known. We report a novel multidentate chelating agent comprising the heterocycles thiadiazole and benzisothiazole. 3-((5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)amino)benzo[d]isothiazole 1,1-dioxide (AL14) was synthesized from cheap saccharin and characterized by different techniques, including single crystal X-ray crystallography. Our studies revealed the efficiency and selectivity of AL14 for the chelation of dissolved Cd(II) (as compared to Cu(II) and Fe(II)). Different spectral changes were observed upon the addition of Cd(II) and Cu(II) during UV-Vis titrations, suggesting different complexation interactions with both metals.

Photoprotective effect of the fucose-containing polysaccharide FucoPol.

This study reports the performance of FucoPol, a fucose-containing bacterial polysaccharide, as a photostable agent with high absorption yield at concentrations as low as 0.02 % (w/v). FucoPol is non-cytotoxic, efficiently protects from UVA and UVB at concentrations of 0.02-2 % (w/v) and 0.2-2 % (w/v), respectively, has over 94 % overall photostability up to 1.5 h of irradiation time. The lowest concentration studied (0.02 %, w/v) shows a sun protection factor (SPF) of 2.61 ± 0.08. The SPF-to-concentration unit ratio showed that FucoPol is about 60-fold more photoprotective than combinations of common organic and inorganic UV filters. In vitro radiation exposure experiments of adhered Vero epithelial and PM1 keratinocytic cells in the presence of 0.25 % (w/v) FucoPol further showed that cell viability was preserved, and delayed radiation-induced cell death was prevented. Overall, FucoPol outperforms common cosmetic biopolymers like xanthan and fucogel. These results are very promising for the development of bio-based sunscreen formulations.

Development of a Cryoprotective Formula Based on the Fucose-Containing Polysaccharide FucoPol.

This study reports the performance of the polysaccharide FucoPol as an enhancer of cryoprotective formulations. FucoPol at a concentration of 0.25% (w/v) was added to several normothermic Dulbecco-derived solutions and hypothermic Euro-Collins, Custodiol-HTK, and Unisol-CV media, substituting some constituents in the latter class to develop FucoPol-based formulations that were tested for their ability to cryopreserve Vero cells. Supplementation yielded post-thaw cell recovery enhancements of at least 70% and averaged at 82%. The FucoPol-supplemented formulations Dulbecco(+)+FP and Unisol-CVS3 achieved cell viabilities capable of competing with the commercial cryogenic formula CryoStor CS5. Particularly in Unisol-CVS3, mannitol, glucose, gluconate, and dextran were all substituted by 0.25% FucoPol, and still, a similar viability was achieved. Multiparametric correlation clustering showed that FucoPol cryoprotection synergizes best with K+, Ca2+, and Cl- in its microenvironment. Component substitution analysis demonstrated that FucoPol acts consistently as a cryoprotectant, an impermeant, and a colloidal stabilizer, providing a combined alternative to glucose, mannitol, gluconate, and dextran, thus highlighting its potential in the development of high-performing cryoprotective formulas.

The Influence of Plasma Composition in the Thermal Cyclic Performance of Yttria-Stabilized Zirconia (8YSZ) Thermal Barrier Coatings (TBCs).

In the combustion chambers of gas turbine engines, ZrO2-8wt.%Y2O3 (YSZ) TBCs are commonly applied by air plasma spray (APS) using Ar-/H2-based plasmas via legacy torches. Alternatively, N2/H2 plasmas could be used with the potential of increasing overall deposition efficiency (DE) and hence reduce the consumption of high-value feedstock powder. Also, by increasing DE, spraying time would be reduced, thereby providing another contribution to bring down production costs. In this work, TBCs were prepared with Ar- and N2-based plasmas with different YSZ powders using Metco 9MB legacy torch. The use of N2-based plasma resulted in higher particle temperature and lower particle velocity values than those provided by the Ar-based plasma. The measured DEs were between 41-43 and 53-60% for the Ar- and N2-based plasmas, respectively. This represents a ~ 40% increase in the DE. The coatings produced with the two different plasmas exhibited equivalent porosity levels ~ 11-13%. On average, the lowest thermal conductivity values were given by a N2-based LD-B YSZ TBC. In the furnace cycle test, the performance of the TBCs prepared with the N2-based plasma was superior to that of the TBCs prepared with the Ar plasma, and also exceeding that of an industrial APS TBC benchmark.

Demonstration of the cryoprotective properties of the fucose-containing polysaccharide FucoPol.

We report the cryoprotective potential of FucoPol, a fucose-containing bacterial exopolysaccharide produced by Enterobacter A47. In vitro cryopreservation assays of Vero, Saos-2, HFFF2 and C2C12 cell lines exposed to a validated non-cytotoxic 2.5 mg/mL FucoPol concentration demonstrated a consistent post-thaw metabolic viability increase. Calorimetric analysis showed a non-colligative, FucoPol concentration-dependent increase of the freezing point (Tf), with minimal change in melting point (Tm). Freezing point variation was corroborated by Polarized Optical Microscopy studies, also showing a reduction of ice crystal dimensions. Its proven shear-thinning behaviour and polyanionicity favour interactivity between the polysaccharide and the water-ice interface, resulting in ice growth inhibition. These findings demonstrate FucoPol's high promise as a bio-based, biodegradable approach to be implemented into cryopreservation formulations.

New endoperoxides highly active in vivo and in vitro against artemisinin-resistant Plasmodium falciparum.

BACKGROUND: The emergence and spread of Plasmodium falciparum resistance to artemisinin-based combination therapy in Southeast Asia prompted the need to develop new endoperoxide-type drugs. METHODS: A chemically diverse library of endoperoxides was designed and synthesized. The compounds were screened for in vitro and in vivo anti-malarial activity using, respectively, the SYBR Green I assay and a mouse model. Ring survival and mature stage survival assays were performed against artemisinin-resistant and artemisinin-sensitive P. falciparum strains. Cytotoxicity was evaluated against mammalian cell lines V79 and HepG2, using the MTT assay. RESULTS: The synthesis and anti-malarial activity of 21 new endoperoxide-derived compounds is reported, where the peroxide pharmacophore is part of a trioxolane (ozonide) or a tetraoxane moiety, flanked by adamantane and a substituted cyclohexyl ring. Eight compounds exhibited sub-micromolar anti-malarial activity (IC50 0.3-71.1 nM), no cross-resistance with artemisinin or quinolone derivatives and negligible cytotoxicity towards mammalian cells. From these, six produced ring stage survival < 1% against the resistant strain IPC5202 and three of them totally suppressed Plasmodium berghei parasitaemia in mice after oral administration. CONCLUSION: The investigated, trioxolane-tetrazole conjugates LC131 and LC136 emerged as potential anti-malarial candidates; they show negligible toxicity towards mammalian cells, ability to kill intra-erythrocytic asexual stages of artemisinin-resistant P. falciparum and capacity to totally suppress P. berghei parasitaemia in mice.

Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics.

The complex fac-[Mo(CO)(3)(histidinate)]Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO.

Inhibition of nitric oxide-stimulated vasorelaxation by carbon monoxide-releasing molecules.

OBJECTIVE: Carbon monoxide (CO) is a weak soluble guanylyl cyclase stimulator, leading to transient increases in cGMP and vasodilation. The aim of the present work was to measure the effect of CO-releasing molecules (CORMs) on the cGMP/nitric oxide (NO) pathway and to evaluate how selected CORMs affect NO-induced vasorelaxation. METHODS AND RESULTS: Incubation of smooth muscle cells with some but not all of the CORMs caused a minor increase in cGMP levels. Concentration-response curves were bell-shaped, with higher CORMs concentrations producing lower increases in cGMP levels. Although exposure of cells to CORM-2 enhanced cGMP formation, we observed that the compound inhibited NO-stimulated cGMP accumulation in cells and NO-stimulated soluble guanylyl cyclase activity that could be reversed by superoxide anion scavengers. Reactive oxygen species generation from CORMs was confirmed using luminol-induced chemiluminescence and electron spin resonance. Furthermore, we observed that NO is scavenged by CORM-2. When used alone CORM-2 relaxed vessels through a cGMP-mediated pathway but attenuated NO donor-stimulated vasorelaxation. CONCLUSION: We conclude that the CORMs examined have context-dependent effects on vessel tone, as they can directly dilate blood vessels, but also block NO-induced vasorelaxation.