Enzymatic Synthesis and Structural Modeling of Bio-Based Oligoesters as an Approach for the Fast Screening of Marine Biodegradation and Ecotoxicity.

Given the widespread use of esters and polyesters in products like cosmetics, fishing nets, lubricants and adhesives, whose specific application(s) may cause their dispersion in open environments, there is a critical need for stringent eco-design criteria based on biodegradability and ecotoxicity evidence. Our approach integrates experimental and computational methods based on short oligomers, offering a screening tool for the rapid identification of sustainable monomers and oligomers, with a special focus on bio-based alternates. We provide insights into the relationships between the chemical structure and properties of bio-based oligomers in terms of biodegradability in marine environments and toxicity in benchmark organisms. The experimental results reveal that the considered aromatic monomers (terephthalic acid and 2,5-furandicarboxylic acid) accumulate under the tested conditions (OECD 306), although some slight biodegradation is observable when the inoculum derives from sites affected by industrial and urban pollution, which suggests that ecosystems adapt to non-natural chemical pollutants. While clean seas are more susceptible to toxic chemical buildup, biotic catalytic activities offer promise for plastic pollution mitigation. Without prejudice to the fact that biodegradability inherently signifies a desirable trait in plastic products, nor that it automatically grants them a sustainable "license", this study is intended to facilitate the rational design of new polymers and materials on the basis of specific uses and applications.

Effects of Regioisomerism on the Antiproliferative Activity of Hydroxystearic Acids on Human Cancer Cell Lines.

A series of regioisomers of the hydroxystearic acid (HSA) was prepared, and the effect of the position of the hydroxyl group along the chain on a panel of human cancer cell lines was investigated. Among the various regioisomers, those carrying the hydroxyl at positions 5, 7, and 9 had growth inhibitor activity against various human tumor cell lines, including CaCo-2, HT29, HeLa, MCF7, PC3, and NLF cells. 10-HSA and 11-HSA showed a very weak effect. 8-HSA did not show inhibitory activity in all cell lines. The biological role of 7-HSA and 9-HSA is widely recognized, while little is known about the effects of 5-HSA. Therefore, the biological effects of 5-HSA in HeLa, HT29, MCF7, and NLF cell lines were investigated using the Livecyte's ptychography technology, which allows correlating changes in proliferation, motility, and morphology as a function of treatment at the same time. 5-HSA not only reduces cell proliferation but also induces changes in cell displacement, directionality, and speed. It is important to characterize the biological effects of 5-HSA, this molecule being an important component of fatty acyl esters of hydroxy fatty acids (FAHFA), a class of endogenous mammalian lipids with noticeable anti-diabetic and anti-inflammatory effects.

Neuroprotective Properties of Cardoon Leaves Extracts against Neurodevelopmental Deficits in an In Vitro Model of Rett Syndrome Depend on the Extraction Method and Harvest Time.

This study investigates the bioactive properties of different extracts of cardoon leaves in rescuing neuronal development arrest in an in vitro model of Rett syndrome (RTT). Samples were obtained from plants harvested at different maturity stages and extracted with two different methodologies, namely Naviglio® and supercritical carbon dioxide (scCO2). While scCO2 extracts more hydrophobic fractions, the Naviglio® method extracts phenolic compounds and less hydrophobic components. Only the scCO2 cardoon leaves extract obtained from plants harvested in spring induced a significant rescue of neuronal atrophy in RTT neurons, while the scCO2 extract from the autumn harvest stimulated dendrite outgrowth in Wild-Type (WT) neurons. The scCO2 extracts were the richest in squalene, 3ß-taraxerol and lupeol, with concentrations in autumn harvest doubling those in spring harvest. The Naviglio® extract was rich in cynaropicrin and exerted a toxic effect at 20 µM on both WT and RTT neurons. When cynaropicrin, squalene, lupeol and 3ß-taraxerol were tested individually, no positive effect was observed, whereas a significant neurotoxicity of cynaropicrin and lupeol was evident. In conclusion, cardoon leaves extracts with high content of hydrophobic bioactive molecules and low cynaropicrin and lupeol concentrations have pharmacological potential to stimulate neuronal development in RTT and WT neurons in vitro.

Soft Nanoonions: A Dynamic Overview onto Catanionic Vesicles Temperature-Driven Transition.

Catanionic vesicles are emerging interesting structures for bioapplications. They self-generate by a pairing of oppositely charged ionic surfactants that assemble into hollow structures. Specifically, the anionic-cationic surfactant pair assumes a double-tailed zwitterionic behavior. In this work, the multilamellar-to-unilamellar thermal transition of several mixed aqueous systems, with a slight excess of the anionic one, were investigated. Interestingly, it was found that the anionic counterion underwent a dissociation as a consequence of a temperature increase, leading to the mentioned thermal transition. The present work proposed the spectroscopic techniques, specifically multinuclear NMR and PGSTE (pulsed gradient stimulated echo), as a key tool to study such systems, with high accuracy and effectiveness, while requiring a small amount of the sample. The results presented herein evidence encouraging perspectives, forecasting the application of the studied vesicular nanoreservoirs, for e.g., drug delivery.

On the Mechanism of Genipin Binding to Primary Amines in Lactose-Modified Chitosan at Neutral pH.

The present manuscript deals with the elucidation of the mechanism of genipin binding by primary amines at neutral pH. UV-VIS and CD measurements both in the presence of oxygen and in oxygen-depleted conditions, combined with computational analyses, led to propose a novel mechanism for the formation of genipin derivatives. The indications collected with chiral and achiral primary amines allowed interpreting the genipin binding to a lactose-modified chitosan (CTL or Chitlac), which is soluble at all pH values. Two types of reaction and their kinetics were found in the presence of oxygen: (i) an interchain reticulation, which involves two genipin molecules and two polysaccharide chains, and (ii) a binding of one genipin molecule to the polymer chain without chain-chain reticulation. The latter evolves in additional interchain cross-links, leading to the formation of the well-known blue iridoid-derivatives.

(R)-10-Hydroxystearic Acid: Crystals vs. Organogel.

The chiral (R)-10-hydroxystearic acid ((R)-10-HSA) is a positional homologue of both (R)-12-HSA and (R)-9-HSA with the OH group in an intermediate position. While (R)-12-HSA is one of the best-known low-molecular-weight organogelators, (R)-9-HSA is not, but it forms crystals in several solvents. With the aim to gain information on the structural role of hydrogen-bonding interactions of the carbinol OH groups, we investigated the behavior of (R)-10-HSA in various solvents. This isomer displays an intermediate behavior between (R)-9 and (R)-12-HSA, producing a stable gel exclusively in paraffin oil, while it crystallizes in other organic solvents. Here, we report the X-ray structure of a single crystal of (R)-10-HSA as well as some structural information on its polymorphism, obtained through X-ray Powder Diffraction (XRPD) and Infrared Spectroscopy (IR). This case study provides new elements to elucidate the structural determinants of the microscopic architectures that lead to the formation of organogels of stearic acid derivatives.

X-Ray Crystal Structures and Organogelator Properties of (R)-9-Hydroxystearic Acid.

(R)-9-hydroxystearic acid, (R)-9-HSA, is a chiral nonracemic hydroxyacid of natural origin possessing interesting properties as an antiproliferative agent against different cancer types. Considering its potential application for medical and pharmaceutical purposes, the structures and rheological properties of (R)-9-HSA were investigated. Oscillatory rheology measurements reveal that (R)-9-HSA gels only paraffin oil, with less efficiency and thermal stability than its positional isomer (R)-12-HSA. Conversely, (R)-9-HSA affords crystals from methanol, acetonitrile, and carbon tetrachloride. The single crystal structures obtained both at 293 K and 100 K show non-centrosymmetric twisted carboxylic acid dimers linked at the midchain OHs into long, unidirectional chains of hydrogen bonds, owing to head-tail ordering of the molecules. Synchrotron X-ray powder diffraction experiments, performed on the solids obtained from different solvents, show the occurrence of polymorphism in paraffin oil and through thermal treatment of the solid from methanol.

Polysaccharide-based networks from homogeneous chitosan-tripolyphosphate hydrogels: synthesis and characterization.

Polysaccharide networks, in the form of hydrogels and dried membranes based on chitosan and on the cross-linker tripolyphosphate (TPP), were developed using a novel approach. TPP was incorporated into chitosan by slow diffusion to favor a controlled gelation. By varying chitosan, TPP, and NaCl concentration, transition from inhomogeneous to homogeneous systems was achieved. Rheology and uniaxial compression tests enabled to identify the best performing hydrogel composition with respect to mechanical properties. FTIR, (31)P NMR, and spectrophotometric methods were used to investigate the interaction chitosan-TPP, the kinetics of phosphates diffusion during the dialysis and the amount of TPP in the hydrogel. A freeze-drying procedure enabled the preparation of soft pliable membranes. The lactate dehydrogenase assay demonstrated the biocompatibility of the membranes toward fibroblasts. Overall, we devised a novel approach to prepare homogeneous macroscopic chitosan/TPP-based biomaterials with tunable mechanical properties and good biocompatibility that show good potential as novel polysaccharide derivatives.

Physical characterization of alginate-Pluronic F127 gel for endoluminal NABDs delivery.

Here we focus the attention on the physical characteristics of a highly biocompatible hydrogel made up of crosslinked alginate and Pluronic F127 (PF127). This is a composite polymeric blend we propose for artery endoluminal delivery of an emerging class of molecules named nucleic acid based drugs (NABDs). The physical characterization of our composite gel, i.e. mesh size distribution and PF127-alginate mutual organization after crosslinking, can significantly determine the NABDs release kinetics. Thus, to explore these aspects, different technical approaches, i.e. rheology, low/high field NMR and TEM, were used. While rheology provided information at the macroscopic and nano-level, the other three approaches gave details at the nano-level. We observe that Pluronic micelles, organizing in cubic ordered domains, generate, upon alginate crosslinking, the formation of meshes (≈ 150 nm) larger than those occurring in a Pluronic-free alginate network (≈ 25 nm). Nevertheless, smaller alginate meshes are still on and can just host un-structured Pluronic micelles and water. Accordingly, the gel structure is quite inhomogeneous, where big meshes (filled by crystalline Pluronic) co-exist with smaller meshes (hosting water and un-structured PF127 micelles). While big meshes offer a considerable hindering action on a diffusing solute, smaller ones represent a sort of free space where solute diffusion is faster. The presence of big and small meshes indicates that drug release may follow a double kinetics characterized by a fast and slow release. Notably, this behavior is considered appropriate for endoluminal drug release to the arterial wall.

Understanding Marine Biodegradation of Bio-Based Oligoesters and Plasticizers.

The study reports the enzymatic synthesis of bio-based oligoesters and chemo-enzymatic processes for obtaining epoxidized bioplasticizers and biolubricants starting from cardoon seed oil. All of the molecules had MW below 1000 g mol-1 and were analyzed in terms of marine biodegradation. The data shed light on the effects of the chemical structure, chemical bond lability, thermal behavior, and water solubility on biodegradation. Moreover, the analysis of the biodegradation of the building blocks that constituted the different bio-based products allowed us to distinguish between different chemical and physicochemical factors. These hints are of major importance for the rational eco-design of new benign bio-based products. Overall, the high lability of ester bonds was confirmed, along with the negligible effect of the presence of epoxy rings on triglyceride structures. The biodegradation data clearly indicated that the monomers/building blocks undergo a much slower process of abiotic or biotic transformations, potentially leading to accumulation. Therefore, the simple analysis of the erosion, hydrolysis, or visual/chemical disappearance of the chemical products or plastic is not sufficient, but ecotoxicity studies on the effects of such small molecules are of major importance. The use of natural feedstocks, such as vegetable seed oils and their derivatives, allows the minimization of these risks, because microorganisms have evolved enzymes and metabolic pathways for processing such natural molecules.

Rational Guidelines for the Two-Step Scalability of Enzymatic Polycondensation: Experimental and Computational Optimization of the Enzymatic Synthesis of Poly(glycerolazelate).

The lipase-catalyzed polycondensation of azelaic acid and glycerol is investigated according to a Design-of-Experiment approach that helps to elucidate the effect of experimental variables on monomer conversion, Mn and regioselectivity of acylation of glycerol. Chemometric analysis shows that after 24 h the reaction proceeds regardless of the presence of the enzyme. Accordingly, the biocatalyst was removed after a first step of synthesis and the chain elongation continued at 80 °C. That allowed the removal of the biocatalyst and the preservation of its activity: pre-requites for efficient applicability at industrial scale. The experimental study, combined with docking-based computational analysis, provides rational guidelines for the optimization of the regioselective acylation of glycerol. The process is scaled up to 73.5 g of monomer. The novelty of the present study is the rigorous control of the reaction conditions and of the integrity of the immobilized biocatalyst, which serve to avoiding any interference of free enzyme or fines released in the reaction mixture. The quantitative analysis of the effect of experimental conditions and the overcoming of some major technical bottlenecks for the scalability of enzymatic polycondensation opens new scenarios for industrial exploitation.

2,5-Furandicarboxaldehyde as a bio-based crosslinking agent replacing glutaraldehyde for covalent enzyme immobilization.

In the quest for a bio-based and safer substitute for glutaraldehyde, we have investigated 2,5 diformylfuran (DFF) as bifunctional crosslinking agent for the covalent immobilization of glucoamylase on amino-functionalized methacrylic resins. Immobilization experiments and systematic comparison with glutaraldehyde at four different concentrations for the activation step showed that DFF leads to comparable enzymatic activities at all tested concentrations. Continuous flow experiment confirms a similar long term stability of the immobilized formulations obtained with the two crosslinkers. The NMR study of DFF in aqueous solution evidenced a much simpler behaviour as compared to glutaraldehyde, since no enolic forms can form and only a mono-hydrated form was observed. Unlike in the case of glutaraldehyde, DFF reacts covalently with the primary amino groups via imine bond formation only. Nevertheless, the stability of the covalent immobilization was confirmed also at acidic pH (4.5), most probably because of the higher stability of the imine bonds formed with the aromatic aldehydes. In terms of toxicity DFF has the advantage of being poorly soluble in water and, more importantly, poorly volatile as compared to glutaraldehyde, which displays severe respiratory toxicity. We have performed preliminary ecotoxicity assays using Aliivibrio fischeri, a marine bacterium, evidencing comparable behaviour (below the toxicity threshold) for both dialdehydes at the tested concentrations.

Resolution of a nonionic surfactant oligomeric mixture by means of DOSY with inverse micelle assistance.

DOSY is a recognized, efficient technique in the analysis of mixtures. It relies on the differences in self-diffusion coefficients, which are determined by the molecular size. Nowadays, efforts are directed towards devising matrices able to interact with the components of the mixture with differential affinity, and therefore capable to interfere with the diffusion processes and to display resolving power towards species of close, or even equal molecular weight, like isomers. Usually, commercial nonionic surfactants are mixtures of oligomeric species, since the head group, which is a short polyoxyehtylene chain, is somewhat polydisperse. The embedment of Igepal CA-520, 5 polyoxyethylene iso-octylphenyl ether, in an inverse microemulsion led to the separation of (1)H signals of the various oligomeric components. This ensued from the differential partitioning between the oil and the surface of the inverse micelles, which depends on the ethyleneoxide number (EON) of the head groups. Thus, it was possible to ascertain that the length distribution of the polyethyleneoxide chains is ingood agreement with the Poisson distribution theoretically predicted for the polymerization of ethylene oxide. The DOSY spectrum contributed to the assignment of the signals and afforded the partition degree, between the two environments, for each individual oligomeric species, providing further insight into nonionic inverse microemulsions, at present widely employed reaction media in the nanotechnological syntheses.

Azelaic Acid: A Bio-Based Building Block for Biodegradable Polymers.

Azelaic acid is a dicarboxylic acid containing nine C atoms, industrially obtained from oleic acid. Besides its important properties and pharmacological applications, as an individual compound, azelaic acid has proved to be a valuable bio-based monomer for the synthesis of biodegradable and sustainable polymers, plasticizers and lubricants. This review discusses the studies and the state of the art in the field of the production of azelaic acid from oleic acid, the chemical and enzymatic synthesis of bio-based oligo and polyester and their properties, including biodegradability and biocompostability.

Soluble HLA-G expression levels and HLA-G/irinotecan association in metastatic colorectal cancer treated with irinotecan-based strategy.

We here explore the soluble Human Leukocyte Antigen-G (sHLA-G) expression level as clinical biomarker in metastatic colorectal cancer (mCRC). To this aim the sHLA-G protein was measured in plasma samples of 40 patients with mCRC treated with the FOLFIRI (irinotecan (CPT-11) plus 5-fluorouracil (5-FU) and leucovorin (LV)) regimen. The results suggest a link between HLA-G levels and irinotecan (CPT-11) pharmacokinetic, leading to hypothesize a molecular interaction between sHLA-G and CPT-11. This interaction was confirmed experimentally by fluorescence spectroscopy. HLA-G is known to exist in a number of polymorphs that affect both the protein expression levels and its peptide-binding cleft. The interaction between HLA-G polymorphs and CPT-11 was explored by means of computational modelling, confirming the hypothesis that CPT-11 could actually target the peptide binding cleft of the most common HLA-G polymorphs.

Design of a Thiosemicarbazide-Functionalized Calix[4]arene Ligand and Related Transition Metal Complexes: Synthesis, Characterization, and Biological Studies.

In this study, we synthesized a new thiosemicarbazide-functionalized calix[4]arene L and its Co2+, Ni2+, Cu2+, and Zn2+ transition metal complexes. For characterization several techniques were employed: Fourier-transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), 13C-NMR, 15N-NMR, correlation spectroscopy (COZY), nuclear Overhauser enhancement spectroscopy (NOESY), electrospray ionization (ESI)-mass spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and elemental analysis. To explore the capability of the thiosemicarbazide function hosted on a calix[4]arene scaffold for growth inhibition of bacteria, fungi, and cancerous tumor cells, a series of biological evaluations were performed. For L, the antimicrobial tests revealed a higher antibacterial activity against gram-positive Bacillus subtilis and a lower activity against gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), whereas the gram-positive Staphylococcus aureus shows resistance. All examined metal derivatives show an enhancement of the antibacterial activity against gram-negative E. coli bacteria, with a more significant improvement for the Ni2+ and Zn2+ complexes. MTT assays showed a considerable in vitro anticancer activity of Co2+, Ni2+, and Cu2+ complexes against Saos-2 bone cancer cell lines. The activity is ascribable to the inorganic ions rather than calixarene ligand. Hemolysis assay results demonstrated that all compounds have high blood compatibility.

Nucleation, reorganization and disassembly of an active network from lactose-modified chitosan mimicking biological matrices.

Developing synthetic materials able to mimic micro- and macrorheological properties of natural networks opens up to novel applications and concepts in materials science. The present contribution describes an active network based on a semi-synthetic polymer, a lactitol-bearing chitosan derivative (Chitlac), and a transient inorganic cross-linker, boric acid. Due to the many and diverse anchoring points for boric acid on the flanking groups of Chitlac, the cross-links constantly break and reform in a highly dynamic fashion. The consequence is a network with unusual non-equilibrium and mechanical properties closely resembling the rheological behavior of natural three-dimensional arrangements and of cytoskeleton. Concepts like network nucleation, reorganization and disassembly are declined in terms of amount of the cross-linker, which acts as a putative motor for remodeling of the network upon application of energy. The out-of-equilibrium and non-linear behavior render the semi-synthetic system of great interest for tissue engineering and for developing in-vitro mimics of natural active matrices.

14N NMR spectra of molecular nitrogen in SDS/pentanol swollen lamellar phases.

14N NMR spectra of air dissolved in lyotropic mesophases are reported. In order to observe the whole spectrum from a molecule in which the quadrupole coupling constant is on the order of a few megahertz, a weak alignment degree with respect to the magnetic field is mandatory. Therefore, dilute lyotropic liquid crystals, namely sodium dodecylsulphate (SDS)/pentanol swollen lamellar phases, were considered. The temperature dependence of the 14N quadrupolar splitting was followed both in the case of oil (either n-dodecane or n-heptane) and brine (a 0.2-M NaBr water solution) swelling. In the former, it paralleled the temperature dependence of the splittings of the alkane deuteria and, in both cases, it was opposite to 23Na quadrupolar splittings. Owing to the higher N2 solubility in hydrocarbons, the 14N NMR spectra provide complementary information to that obtained by means of the quadrupolar nuclei of water and hydrophilic solutes.

Stabilization of unilamellar catanionic vesicles induced by ß-cyclodextrins: A strategy for a tunable drug delivery depot.

The limited stability of catanionic vesicles has discouraged their wide use for encapsulation and controlled release of active substances. Their structure can easily break down to form lamellar phases, micelles or rearrange into multilamellar vesicles, as a consequence of small changes in their composition. However, despite the limited stability, catanionic vesicles possess an attractive architecture, which is able to efficiently encapsulate both hydrophobic and hydrophilic molecules. Therefore, improving the stability of the vesicles, as well as the control on unilamellar structures, are prerequisites for their wider application range. This study focuses on the impact of ß-cyclodextrins for the stabilization of SDS/CTAB catanionic vesicles. Molar ratio and sample preparation procedures have been investigated to evaluate the temperature stability of catanionic vesicles. Diffusion and spectroscopic techniques evidenced that when ß-cyclodextrins are added, unilamellar structures are stabilized above the multilamellar-unilamellar vesicles critical temperature. The results evidence encouraging perspectives for the use of vesicular nanoreservoirs for drug depot applications.

The role played by the molecular weight and acetylation degree in modulating the stiffness and elasticity of chitosan gels.

A broad library of chitosans was produced varying the molecular weight and the fraction of acetylated units, FA. The produced chitosans were used for the formation of wall-to-wall cylindrical gels through a controlled external gelation using tripolyphosphate (TPP) as cross-linker. The resulting gels were analyzed by rheometry. Viscosity average degree of polymerization (DPv¯) > 152 was shown to be required for the formation of stable gels. Both gel stiffness and gel rupture strength were proportional to the molecular weight regardless of the applied deformation. Increasing acetylation produced a marked reduction of the shear modulus, but, in parallel, switched the networks from rigid and brittle to weak and elastic. Intriguingly, gels made of chitosan with FA = 0.37 displayed notable elasticity, i.e. up to 90% of applied strain falls into linear regime. These findings suggest that the frequency of glucosamine (D unit) and N-acetyl-glucosamine (A unit) contribute to a subtle structure-property relationship in chitosan-TPP gels.