Long chain capsaicin analogues synthetized by CALB-CLEAs show cytotoxicity on glioblastoma cell lines.

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives. KEY POINTS: • This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs. • The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil. • Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells.

Computational insights of the molecular recognition between volatile molecules and odorant binding proteins from the red palm weevil Rhynchophorus ferrugineus.

The red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) is one of the most harmful pests for palm trees, causing serious economic damage worldwide. The present work aims to model and study the 3D structures of highly expressed odorant binding proteins from R. ferrugineus (RferOBPs) and identify possible binding modes and ligand release mechanism by docking and molecular dynamics. Highly confident 3D structures of a total of 11 odorant binding proteins (OBPs) were obtained with AlphaFold2. All 3D RferOBPs modeled structures displayed six characteristic α-helices, except for RfeOBP7 and RfeOBP10, which had an extra terminal α-helix. Among the eleven modeled RferOBPs, RferOBP4 was highly expressed in the antennae and subsequently selected for further analyses. Molecular docking analyses demonstrated that ferruginol, α-pinene, DEET, and picaridin can favorably bind the RferOBP4 cavity with low affinity energies. Molecular dynamic simulations of RferOBP4 bound to ferruginol at different pH values showed that low pH environments dictate a structural change into an apo-state that modifies the number of tunnels where the ligand can coexist, further triggering ligand release by a pH-dependent mechanism. This is the first report concerning the modelling and study of ligand binding modes and release mechanism of R. ferrugineus OBPs.Communicated by Ramaswamy H. Sarma.

Structural Behavior of Amphiphilic Triblock Copolymer P104/Water System.

A detailed study of the different structural transitions of the triblock copolymer PEO27-PPO61-PEO27 (P104) in water, in the dilute and semi-dilute regions, is addressed here as a function of temperature and P104 concentration (CP104) by mean of complimentary methods: viscosimetry, densimetry, dynamic light scattering, turbidimetry, polarized microscopy, and rheometry. The hydration profile was calculated through density and sound velocity measurements. It was possible to identify the regions where monomers exist, spherical micelle formation, elongated cylindrical micelles formation, clouding points, and liquid crystalline behavior. We report a partial phase diagram including information for P104 concentrations from 1 × 10-4 to 90 wt.% and temperatures from 20 to 75 °C that will be helpful for further interaction studies with hydrophobic molecules or active principles for drug delivery.

Development of functional hybrid scaffolds for wound healing applications.

Hybrid hydrogels composed of chitosan (CS) and hyaluronic acid (HA) and collagen (Coll) were prepared by polyelectrolyte complex self-assembly. These scaffolds displayed a good intermingling of the polymeric chains, with porosities above 80% and good interconnected structures with pore sizes lying between 30-115 µm. The ionic interactions between CS and HA make the scaffolds have larger storage modulus and longer LVR regions than their pure counterparts. Both quantities progressively decrease as the HA and Coll concentrations in the formulation rise. These hybrid hydrogels showed good swelling extents from ca. 420 to ca. 690% and suitable resistance to enzymatic degradation, which was slightly lower for scaffolds containing CS to larger extents or Coll in the formulation. All scaffolds were largely cytocompatible and allowed the proliferation of both mouse fibroblast and human keratinocytes with their infiltration inside, thus becoming optimal matrices for intended tissue engineering applications as well as transdermal drug delivery depots.

Topical Pirfenidone-Loaded Liposomes Ophthalmic Formulation Reduces Haze Development after Corneal Alkali Burn in Mice.

Corneal chemical burns (CCBs) frequently result in corneal fibrosis or haze, an opacity of the cornea that obstructs vision and induces corneal blindness. Diverse strategies have been employed to prevent or reduce CCB-related corneal haze. In this study, we evaluated the physicochemical characteristics and biologic effects of a topical pirfenidone (PFD)-loaded liposomal formulation (PL) on a corneal alkali burn mice model. We found that PL was appropriate for ocular application due to its physiologic tear pH, osmolarity and viscosity suitable for topical ophthalmic use. Regarding its therapeutic activity, PL-treated mice had significantly reduced haze size and density, corneal edema, corneal thickness, and corneal inflammatory infiltration, in contrast to PFD in aqueous solution (p < 0.01). Importantly, the antifibrotic activity of PL (reduction of corneal haze) was associated with modulation of transforming growth factor (TGF)-ß and Interleukin (IL)-1ß genes. PL suppressed TGF-ß expression and restored normal IL-1ß expression in corneal tissue more efficiently in contrast to PFD in aqueous solution. In conclusion, PFD showed essential anti-inflammatory and anti-fibrotic effects in the treatment of alkali burns. Noteworthy, a new formulation of PFD-loaded liposomes remarkably improved these effects, standing out as a promising treatment for corneal haze.

Mechanical and Physicochemical Properties of 3D-Printed Agave Fibers/Poly(lactic) Acid Biocomposites.

In order to provide a second economic life to agave fibers, an important waste material from the production of tequila, filaments based on polylactic acid (PLA) were filled with agave fibers (0, 3, 5, 10 wt%), and further utilized to produce biocomposites by fused deposition modeling (FDM)-based 3D printing at two raster angles (-45°/45° and 0°/90°). Differential scanning calorimetry, water uptake, density variation, morphology, and composting of the biocomposites were studied. The mechanical properties of the biocomposites (tensile, flexural, and Charpy impact properties) were determined following ASTM international norms. The addition of agave fibers to the filaments increased the crystallinity value from 23.7 to 44.1%. However, the fibers generated porous structures with a higher content of open cells and lower apparent densities than neat PLA pieces. The printing angle had a low significant effect on flexural and tensile properties, but directly affected the morphology of the printed biocomposites, positively influenced the impact strength, and slightly improved the absorption values for biocomposites printed at -45°/45°. Overall, increasing the concentrations of agave fibers had a detrimental effect on the mechanical properties of the biocomposites. The disintegration of the biocomposites under simulated composting conditions was slowed 1.6-fold with the addition of agave fibers, compared to neat PLA.

Optimization of the Obtaining of Cellulose Nanocrystals from Agave tequilana Weber Var. Azul Bagasse by Acid Hydrolysis.

A multilevel factorial design of 23 with 12 experiments was developed for the preparation of cellulose nanocrystals (CNC) from Agave tequilana Weber var. Azul bagasse, an agro-industrial waste from tequila production. The studied parameters were acid type (H2SO4 and HCl), acid concentration (60 and 65 wt% for H2SO4, 2 and 8N for HCl) temperature (40 and 60 °C for H2SO4, 50 and 90 °C for HCl), and hydrolysis time (40, 55 and 70 min for H2SO4; and 30, 115 and 200 min for HCl). The obtained CNC were physical and chemically characterized using dynamic light scattering (DLS), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XDR) techniques. The maximum CNC yield was 90 and 96% for HCL and H2SO4, respectively, and the crystallinity values ranged from 88-91%. The size and morphology of A. tequilana CNC strongly depends on the acid type and hydrolysis time. The shortest CNC obtained with H2SO4 (65 wt%, 40 °C, and 70 min) had a length of 137 ± 68 nm, width 33 ± 7 nm, and height 9.1 nm, whereas the shortest CNC obtained with HCl (2 N, 50 °C and 30 min) had a length of 216 ± 73 nm, width 69 ± 17 nm, and height 8.9 nm. In general, the obtained CNC had an ellipsoidal shape, whereas CNC prepared from H2SO4 were shorter and thinner than those obtained with HCl. The total sulfate group content of CNC obtained with H2SO4 increased with time, temperature, and acid concentration, exhibiting an exponential behavior of CSG=aebt.

Rotational Molding of Poly(Lactic Acid)/Polyethylene Blends: Effects of the Mixing Strategy on the Physical and Mechanical Properties.

In this study, blends of poly(lactic acid) (PLA)/linear medium density polyethylene (LMDPE) at different weight ratios were prepared by rotational molding. Two mixing strategies were used to evaluate the effect of phase dispersion on the physical and mechanical properties: (i) Dry-blending (DB) using a high shear mixer, and (ii) melt-blending (MB) using a twin-screw extruder. Thermal, morphological, and mechanical analyses were performed on the neat polymers and their blends. The thermal analysis was completed by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), and the blends prepared by MB had lower thermal stability than the ones prepared via DB due to some thermo-oxidative degradation through the double thermal process (extrusion and rotomolding). The morphology of the rotomolded parts showed that DB generated larger particle sizes (around 500 µm) compared to MB (around 5 µm) due to the shear and elongational stresses applied during extrusion. The tensile and flexural properties of the rotomolded parts combined the PLA stiffness with the LMDPE toughness independent of the blending technique. Neat PLA presented increments in tensile strength (54%) and flexural strength (111%) for DB compared with MB. A synergistic effect in impact strength was observed in blends with 12 and 25 wt. % of PLA prepared by DB.

Adsorption and Interaction of Bovine Serum Albumin and Pluronic P103 Triblock Copolymer on a Gold Electrode: Double-Layer Capacitance Measurements.

The interactions of proteins and other molecules and their adsorption onto substrates is a fascinating topic that has been applied to surface technologies, biosensors, corrosion studies, biotechnologies, and other fields. The success of these applications requires a previous characterization using some analytical techniques that, ordinarily, are not electrochemical. This work proposes analyzing the variation of the double-layer capacitance obtained through impedance electrochemical spectroscopy as an alternative strategy to show evidence of the interactions between proteins and triblock copolymers. The proposal is supported through the study of the interaction and adsorption of bovine serum albumin (BSA) and a commercial triblock copolymer (P103) in phosphate buffer on a gold electrode. The double-layer capacitance and the apparent interface thickness vs polarization potential curves as well as the potential of zero charge for pure P103 (0.6 wt %, corresponding to 6 g L-1), pure BSA (3 mg mL-1), and P103-BSA solutions (0.6 wt % and 3 mg mL-1, respectively) are sensitive enough to show not only the interaction and the adsorption of the species but also the polarization potential where these interactions are taking place. A qualitative and quantitative analysis concerning the double-layer capacitance behavior is given. The significance and impact of this work is also presented.

Cross-linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil, a nonpungent capsaicin analogue.

Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L-1 of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L-1 of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.

Disulfide-modified antigen for detection of celiac disease-associated anti-tissue transglutaminase autoantibodies.

A simple and rapid immunosensor for the determination of the celiac disease-related antibody, anti-tissue transglutaminase, was investigated. The antigenic protein tissue transglutaminase was chemically modified, introducing disulfide groups through different moieties of the molecule (amine, carboxylic, and hydroxyl groups), self-assembled on gold surfaces, and used for the detection of IgA and IgG autoantibodies. The modified proteins were evaluated using enzyme-linked immunosorbent assay and surface plasmon resonance, which showed that only introduction of the disulfide groups through amine moieties in the tissue transglutaminase preserved its antigenic properties. The disulfide-modified antigen was co-immobilized via chemisorption with a poly(ethylene glycol) alkanethiol on gold electrodes. The modified electrodes were then exposed to IgA anti-tissue transglutaminase antibodies and subsequently to horseradish peroxidase-labeled anti-idiotypic antibodies, achieving a detection limit of 260 ng ml-1. Immunosensor performance in the presence of complex matrixes, including clinically relevant serum reference solutions and real patient samples, was evaluated. The introduction of disulfides in the antigenic protein enabled a simple and convenient one-step surface immobilization procedure involving only spontaneous gold-thiol covalent binding. Complete amperometric assay time was 30 min.

Amperometric immunosensor for the determination of IgA deficiency in human serum samples.

An electrochemical immunosensor for the detection of human IgA deficiency in real human blood serum has been developed. The performance of the immunosensor presents a large but sensitive dynamic range that allows the determination of non-deficient IgA levels (>70 µg/mL) as well as of severe IgA deficiencies (0.5-5.0 µg/mL). The assay architecture involves the immobilisation of a coating antibody on an electrode surface using carboxylic-ended bipodal alkane-thiol self-assembled monolayers (SAMs). The long chain bipodal SAM presents intercalated poly(ethylenglycol) groups that confer the immunosensor the ability to retain its optimum performance in very complex matrices and serum with negligible non-specific adsorption phenomena. Amperometric optimisation of the assay resulted in limits of detection of 142 ng/mL in just 30 min total assay time. Real patients' serum samples were analysed using the developed electrochemical immunosensor demonstrating an excellent correlation in terms of sensitivity and reproducibility compared with standard enzyme linked immunosorbent assays (ELISA).