Bioconjugation of Vegetable Oils with UV Absorbers: New Approach in Skin Photoprotection.

We reported the tunable synthesis of new vegetable oil-UV filter bioconjugates using sea buckthorn oil (SBO) and p-methoxycinnamic acid (p-MCA) as an alternative to the common UV filter, ethylhexyl-p-methoxycinnamate (octinoxate). The synthetic strategy is based on the sustainable ring-opening reaction of epoxidized SBO with p-MCA in heterogenous catalysis in eco-friendly solvents. The amount of UV-absorptive moieties grafted on the triglyceride backbone is controlled by different epoxidation degrees as determined by NMR spectroscopy. The performance of the new UV-absorber bioconjugates was assessed by in vitro sun protection factor (SPF) measurements after inclusion in SBO-ethylcellulose (EC) oleogels and comparison with the SPF value of the SBO-EC-octinoxate oleogel with equivalent p-MCA acid moieties (10% wt/wt). The concentration obtained for the SBO-EC oleogel formulated with the bioconjugate with the lowest degree of functionalization, namely 55%, represents 45% of the SPF determined for the SBO-EC-octinoxate oleogel, regardless of the concentration of measured solutions. The new concept of vegetable oil-UV-absorber bioconjugates has potential UV-B photoprotective properties when included in oleogel formulations and deserves further investigation of their properties and stability including association with UV-A absorbers, respectively.

3D Printable Composite Biomaterials Based on GelMA and Hydroxyapatite Powders Doped with Cerium Ions for Bone Tissue Regeneration.

The main objective was to produce 3D printable hydrogels based on GelMA and hydroxyapatite doped with cerium ions with potential application in bone regeneration. The first part of the study regards the substitution of Ca2+ ions from hydroxyapatite structure with cerium ions (Ca10-xCex(PO4)6(OH)2, xCe = 0.1, 0.3, 0.5). The second part followed the selection of the optimal concentration of HAp doped, which will ensure GelMA-based scaffolds with good biocompatibility, viability and cell proliferation. The third part aimed to select the optimal concentrations of GelMA for the 3D printing process (20%, 30% and 35%). In vitro biological assessment presented the highest level of cell viability and proliferation potency of GelMA-HC5 composites, along with a low cytotoxic potential, highlighting the beneficial effects of cerium on cell growth, also supported by Live/Dead results. According to the 3D printing experiments, the 30% GelMA enriched with HC5 was able to generate 3D scaffolds with high structural integrity and homogeneity, showing the highest suitability for the 3D printing process. The osteogenic differentiation experiments confirmed the ability of 30% GelMA-3% HC5 scaffold to support and efficiently maintain the osteogenesis process. Based on the results, 30% GelMA-3% HC5 3D printed scaffolds could be considered as biomaterials with suitable characteristics for application in bone tissue engineering.

Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions.

Protein and peptide interactions are characterized in the liquid state by multidimensional NMR spectroscopy experiments, which can take hours to record. We show that starting from hyperpolarized HDO, two-dimensional (2D) proton correlation maps of a peptide, either free in solution or interacting with liposomes, can be acquired in less than 60 s. In standard 2D NMR spectroscopy without hyperpolarization, the acquisition time required for similar spectral correlations is on the order of hours. This hyperpolarized experiment enables the identification of amino acids featuring solvent-interacting hydrogens and provides fast spectroscopic analysis of peptide conformers. Sensitivity-enhanced 2D proton correlation spectroscopy is a useful and straightforward tool for biochemistry and structural biology, as it does not recur to nitrogen-15 or carbon-13 isotope enrichment.

Hyperpolarised NMR to follow water proton transport through membrane channels via exchange with biomolecules.

Water uptake in vesicles and the subsequent exchange between water protons and amide -NH protons in amino acids can be followed by a new, highly sensitive, type of magnetic resonance spectroscopy: dynamic nuclear polarisation (DNP)-enhanced NMR in the liquid state. Water hydrogen atoms are detected prior to and after their transfer to molecular sites in peptides and proteins featuring highly-accessible proton-exchangeable groups, as is the case for the -NH groups of intrinsically disordered proteins. The detected rates for amide proton-water proton exchange can be modulated by membrane-crossing rates, when a membrane channel is interposed. We hyperpolarised water proton spins via dynamic nuclear polarisation followed by sample dissolution (d-DNP) and transferred the created polarisation to -NH groups with high solvent accessibility in an intrinsically disordered protein domain. This domain is the membrane anchor of c-Src kinase, whose activity controls cell proliferation. The hindrance of effective water proton transfer rate constants observed in free solvent when a membrane-crossing step is involved is discussed. This study aims to assess the feasibility of recently-introduced hyperpolarised (DNP-enhanced) NMR to assess water membrane crossing dynamics.

Fabrication of k-Carrageenan/Alginate/Carboxymethyl Cellulose basedScaffolds via 3D Printing for Potential Biomedical Applications.

Three-dimensional (3D) printing technology was able to generate great attention because of its unique methodology and for its major potential to manufacture detailed and customizable scaffolds in terms of size, shape and pore structure in fields like medicine, pharmaceutics and food. This study aims to fabricate an ink entirely composed of natural polymers, alginate, k-carrageenan and carboxymethyl cellulose (AkCMC). Extrusion-based 3D printing was used to obtain scaffolds based on a crosslinked interpenetrating polymer network from the alginate, k-carrageenan, carboxymethyl cellulose and glutaraldehide formulation using CaCl2, KCl and glutaraldehyde in various concentrations of acetic acid. The stabile bonding of the crosslinked scaffolds was assessed using infrared spectroscopy (FT-IR) as well as swelling, degradation and mechanical investigations. Moreover, morphology analysis (µCT and SEM) confirmed the 3D printed samples' porous structure. In the AkCMC-GA objects crosslinked with the biggest acetic acid concentration, the values of pores and walls are the highest, at 3.9 × 10-2 µm-1. Additionally, this research proves the encapsulation of vitamin B1 via FT-IR and UV-Vis spectroscopy. The highest encapsulation efficiency of vitamin B1 was registered for the AkCMC-GA samples crosslinked with the maximum acetic acid concentration. The kinetic release of the vitamin was evaluated by UV-Vis spectroscopy. Based on the results of these experiments, 3D printed constructs using AkCMC-GA ink could be used for soft tissue engineering applications and also for vitamin B1 encapsulation.

Three-Dimensional-Printed Sodium Alginate and k-Carrageenan-Based Scaffolds with Potential Biomedical Applications.

This work reports the development of a marine-derived polysaccharide formulation based on k-Carrageenan and sodium alginate in order to produce a novel scaffold for engineering applications. The viscoelastic properties of the bicomponent inks were assessed via rheological tests prior to 3D printing. Compositions with different weight ratios between the two polymers, without any crosslinker, were subjected to 3D printing for the first time, to the best of our knowledge, and the fabrication parameters were optimized to ensure a controlled architecture. Crosslinking of the 3D-printed scaffolds was performed in the presence of a chloride mixture (CaCl2:KCl = 1:1; v/v) of different concentrations. The efficiency of the crosslinking protocol was evaluated in terms of swelling behavior and mechanical properties. The swelling behavior indicated a decrease in the swelling degree when the concentration of the crosslinking agent was increased. These results are consistent with the nanoindentation measurements and the results of the macro-scale tests. Moreover, morphology analysis was also used to determine the pore size of the samples upon freeze-drying and the uniformity and micro-architectural characteristics of the scaffolds. Overall, the registered results indicated that the bicomponent ink, Alg/kCG = 1:1 may exhibit potential for tissue-engineering applications.

Electrospun Nanofibrous Membranes Based on Citric Acid-Functionalized Chitosan Containing rGO-TEPA with Potential Application in Wound Dressings.

The present research work is focused on the design and investigation of electrospun composite membranes based on citric acid-functionalized chitosan (CsA) containing reduced graphene oxide-tetraethylene pentamine (CsA/rGO-TEPA) as materials with opportune bio-properties for applications in wound dressings. The covalent functionalization of chitosan (CS) with citric acid (CA) was achieved through the EDC/NHS coupling system and was checked by 1H-NMR spectroscopy and FTIR spectrometry. The mixtures to be electrospun were formulated by adding three concentrations of rGO-TEPA into the 1/1 (w/w) CsA/poly (ethylene oxide) (PEO) solution. The effect of rGO-TEPA concentration on the morphology, wettability, thermal stability, cytocompatibility, cytotoxicity, and anti-biofilm activity of the nanofibrous membranes was extensively investigated. FTIR and Raman results confirmed the covalent and non-covalent interactions that appeared between the system's compounds, and the exfoliation of rGO-TEPA sheets within the CsA in the presence of PEO (CsA/P) polymer matrix, respectively. SEM analysis emphasized the nanofibrous architecture of membranes and the presence of rGO-TEPA sheets entrapped into the CsA nanofiber structure. The MTT cellular viability assay showed a good cytocompatibility with the highest level of cell development and proliferation registered for the CsA/P composite nanofibrous membrane with 0.250 wt.% rGO-TEPA. The designed nanofibrous membranes could have potential applications in wound dressings, given that they showed a good anti-biofilm activity against Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacterial strains.

Macrophage-targeted mannose-decorated PLGA-vegetable oil hybrid nanoparticles loaded with anti-inflammatory agents.

This work pledge to extend the therapeutic windows of hybrid nanoparticulate systems by engineering mannose-decorated hybrid nanoparticles based on poly lactic-co-glycolic acid (PLGA) and vegetable oil for efficient delivery of two lipophilic anti-inflammatory therapeutics (Celecoxib-CL and Indomethacin-IMC) to macrophages. The mannose surface modification of nanoparticles is achieved via O-palmitoyl-mannose spacer during the emulsification and nanoparticles assembly process. The impact of targeting motif on the hydrodynamic features (RH, PdI), stability (ζ-potential), drug encapsulation efficiency (DEE) is thoroughly investigated. Besides, the in vitro biocompatibility (MTT, LDH) and susceptibility of mannose-decorated formulations to macrophage as well their immunomodulatory activity (ELISA) are also evaluated. The monomodal distributed mannose-decorated nanoparticles are in the range of nanometric size (RH < 115 nm) with PdI < 0.20 and good encapsulation efficiency (DEE = 46.15% for CL and 76.20% for IMC). The quantitative investigation of macrophage uptake shows a 2-fold increase in fluorescence (RFU) of cells treated with mannose-decorated formulations as compared to non-decorated ones (p < 0.001) suggesting an enhanced cell uptake respectively improved macrophage targeting while the results of ELISA experiments suggest the potential immunomodulatory properties of the designed mannose-decorated hybrid formulations.

Saponification Value of Fats and Oils as Determined from 1H-NMR Data: The Case of Dairy Fats.

The saponification value of fats and oils is one of the most common quality indices, reflecting the mean molecular weight of the constituting triacylglycerols. Proton nuclear magnetic resonance (1H-NMR) spectra of fats and oils display specific resonances for the protons from the structural patterns of the triacylglycerols (i.e., the glycerol backbone), methylene (-CH2-) groups, double bonds (-CH=CH-) and the terminal methyl (-CH3) group from the three fatty acyl chains. Consequently, chemometric equations based on the integral values of the 1H-NMR resonances allow for the calculation of the mean molecular weight of triacylglycerol species, leading to the determination of the number of moles of triacylglycerol species per 1 g of fat and eventually to the calculation of the saponification value (SV), expressed as mg KOH/g of fat. The algorithm was verified on a series of binary mixtures of tributyrin (TB) and vegetable oils (i.e., soybean and rapeseed oils) in various ratios, ensuring a wide range of SV. Compared to the conventional technique for SV determination (ISO 3657:2013) based on titration, the obtained 1H-NMR-based saponification values differed by a mean percent deviation of 3%, suggesting the new method is a convenient and rapid alternate approach. Moreover, compared to other reported methods of determining the SV from spectroscopic data, this method is not based on regression equations and, consequently, does not require calibration from a database, as the SV is computed directly and independently from the 1H-NMR spectrum of a given oil/fat sample.

A Real-Life Reproducibility Assessment for NMR Metabolomics.

Nuclear magnetic resonance (NMR) metabolomics is currently popular enough to attract both specialized and non-specialized NMR groups involving both analytical trained personnel and newcomers, including undergraduate students. Recent interlaboratory studies performed by established NMR metabolomics groups demonstrated high reproducibility of the state-of-the-art NMR equipment and SOPs. There is, however, no assessment of NMR reproducibility when mixing both analytical experts and newcomers. An interlaboratory assessment of NMR quantitation reproducibility was performed using two NMR instruments belonging to different laboratories and involving several operators with different backgrounds and metabolomics expertise for the purpose of assessing the limiting factors for data reproducibility in a multipurpose NMR environment. The variability induced by the operator, automatic pipettes, NMR tubes and NMR instruments was evaluated in order to assess the limiting factors for quantitation reproducibility. The results estimated the expected reproducibility data in a real-life multipurpose NMR laboratory to a maximum 4% variability, demonstrating that the current NMR equipment and SOPs may compensate some of the operator-induced variability.

3D-Printed Gelatin Methacryloyl-Based Scaffolds with Potential Application in Tissue Engineering.

The development of materials for 3D printing adapted for tissue engineering represents one of the main concerns nowadays. Our aim was to obtain suitable 3D-printed scaffolds based on methacrylated gelatin (GelMA). In this respect, three degrees of GelMA methacrylation, three different concentrations of GelMA (10%, 20%, and 30%), and also two concentrations of photoinitiator (I-2959) (0.5% and 1%) were explored to develop proper GelMA hydrogel ink formulations to be used in the 3D printing process. Afterward, all these GelMA hydrogel-based inks/3D-printed scaffolds were characterized structurally, mechanically, and morphologically. The presence of methacryloyl groups bounded to the surface of GelMA was confirmed by FTIR and 1H-NMR analyses. The methacrylation degree influenced the value of the isoelectric point that decreased with the GelMA methacrylation degree. A greater concentration of photoinitiator influenced the hydrophilicity of the polymer as proved using contact angle and swelling studies because of the new bonds resulting after the photocrosslinking stage. According to the mechanical tests, better mechanical properties were obtained in the presence of the 1% initiator. Circular dichroism analyses demonstrated that the secondary structure of gelatin remained unaffected during the methacrylation process, thus being suitable for biological applications.

5-Aminosalicylic Acid Loaded Chitosan-Carrageenan Hydrogel Beads with Potential Application for the Treatment of Inflammatory Bowel Disease.

The aim of our work is to prepare mucoadhesive particles with biopolymers and 5-Aminosalicylic acid (5ASA) using the ionotropic gelation technique to ensure a controlled drug release at the colon level with potential applications in the treatment of intestinal bowel disease (IBD). The preparation of particles through the crosslinking of Chitosan (CS) with sodium tripolyphosphate (TPP) using different mass ratios and the influence of the k-Carrageenan (kCG) layer were studied. UV-VIS spectrometry was employed to assess encapsulation efficiency and drug release profile of 5ASA. The particles were investigated using FT-IR spectrometry for chemical characterization and the DLS results highlighted a monodisperse particle size distribution. The morphology of the polymeric beads was investigated using micro-computer tomography (µCT) and Scanning Electron Microscopy (SEM). Particles based on Chitosan and k-Carrageenan were able to incorporate and preserve 5ASA in an acidic and alkaline medium. The 5ASA loaded polymeric particles obtained after immersion for 1 h in kCG solution exhibited the lowest release rate in pH = 1.2. Biocompatibility studies performed on all of the particles displayed a good viability for the CCD 841 CoN cells and low cytotoxicity. All of the results have shown that these new biomaterials could be a versatile platform of targeted carriers with potential applications in inflammatory bowel disease treatment.

Synthesis, density functional theory study and in vitro antimicrobial evaluation of new benzimidazole Mannich bases.

The tri-component synthesis of novel chiral benzimidazole Mannich bases, by reaction between benzimidazole, aqueous 30% formaldehyde and an amine, the biological evaluation and DFT studies of the new compounds are reported here. The 1H-NMR, 13C-NMR, FTIR spectra and elemental analysis confirm the structures of the new compounds. All synthesized compounds were screened by qualitative and quantitative methods for their in vitro antibacterial activity against 4 bacterial strains. DFT studies were accomplished using GAMESS 2012 software and HOMO-LUMO analysis allowed the calculation of electronic and structural parameters of the chiral Mannich bases. The geometry of 1-methylpiperazine, the cumulated Mullikan atomic charges of the two heteroatoms and of the methyl, and the value of the global electrophilicity index (ω = 0.0527) of the M-1 molecule is correlated with its good antimicrobial activity. It was found that the presence of saturated heterocycles from the amine molecule, 1-methyl piperazine and morpholine, respectively, contributes to an increased biological activity, compared to aromatic amino analogs, diphenylamino-, 4-nitroamino- and 4-aminobenzoic acid. The planarity of the molecules, specific bond lengths and localization of HOMO-LUMO orbitals is responsible for the best biological activities of the compounds.

Water hydrogen uptake in biomolecules detected via nuclear magnetic phosphorescence.

We introduce a new symmetry-based method for structural investigations of areas surrounding water-exchanging hydrogens in biomolecules by liquid-state nuclear magnetic resonance spectroscopy. Native structures of peptides and proteins can be solved by NMR with fair resolution, with the notable exception of labile hydrogen sites. The reason why biomolecular structures often remain elusive around exchangeable protons is that the dynamics of their exchange with the solvent hampers the observation of their signals. The new spectroscopic method we report allows to locate water-originating hydrogens in peptides and proteins via their effect on nuclear magnetic transitions similar to electronic phosphorescence, long-lived coherences. The sign of long-lived coherences excited in coupled protons can be switched by the experimenter. The different effect of water-exchanging hydrogens on long-lived coherences with opposed signs allows to pinpoint the position of these labile hydrogen atoms in the molecular framework of peptides and proteins.

A novel profluorescent paramagnetic diaza-crown ether: synthesis, characterization and alkaline metal-ion complexation.

Starting from Kryptofix 22 two different branches were covalently attached through the nitrogen atoms, one containing a fluorescent moiety and the other the stable free radical TEMPO. The novel derivative exhibits fluorescence and paramagnetic properties, while the diaza-crown part ensures the affinity for alkaline metal-ions.

Long-lived states detect interactions between small molecules and diamagnetic metal ions.

Long-lived states of nuclear spin order were used for the first time to probe interactions between molecules and diamagnetic metal ions. Proton spin states with lifetimes twice as long as the spin-lattice relaxation time constants of the same nuclei were promoted on the methoxyphenyl and tolyl substituents of a 1,3,4-oxadiazole derivative. The transient interaction of this oxadiazole derivative with silver(I) ions significantly speeds up the relaxation rate constants of proton long-lived states. The interactions between silver and organic compounds lead to the formation of coordination polymers that can be used for the preparation of bio-compatible materials.