Synthesis, characterization, and in vitro-in ovo toxicological screening of silibinin fatty acids conjugates as prodrugs with potential biomedical applications.

Silibinin (SIL), the most active phytocompound from Silybum marianum (L.), exerts many biological effects but has low stability and bioavailability. To overcome these drawbacks, the current research proposed the synthesis of silibilin oleate (SIL-O) and silibilin linoleate (SIL-L) derivatives as prodrugs with potentially optimized properties for biomedical applications, and the establishment of their in vitro-in ovo safety profiles. The physicochemical characterization of the obtained compounds using density functional theory (DFT) calculations, and Raman and 1H liquid-state nuclear magnetic resonance (NMR) spectroscopy confirmed the formation of SIL-O and SIL-L complexes. Computational predictions revealed that these lipophilic derivatives present a lower drug-likeness score (-29.96 for SIL-O and -23.55 for SIL-L) compared to SIL, but an overall positive drug score (0.07) and no risk for severe adverse effects. SIL-O and SIL-L showed no cytotoxicity or impairment in cell migration at low concentrations, but at the highest concentration (100 µM), they displayed distinct toxicological profiles. SIL-L was more cytotoxic (on cardiomyoblasts - H9c2(2-1), hepatocytes - HepaRG, and keratinocytes - HaCaT) than SIL-O or SIL, significantly inhibiting cell viability (< 60%), altering cellular morphology, reducing cell confluence (< 70%), and inducing prominent apoptotic-like nuclear features. At the concentration of 100 µM, SIL-O presented an irritation score (IS) of 0.61, indicating a lack of irritant effect on the chorioallantoic membrane (CAM), while SIL-L was classified as a slight irritant with an IS of 1.99. These findings outline a more favorable in vitro and in ovo biocompatibility for SIL-O compared to SIL-L, whose applications are dosage-limited due to potential toxicity.

pH-Dependent Behavior of Novel 5-FU Delivery System in Environmental Conditions Comparable to the Gastro-Intestinal Tract.

A biogenic carrier for 5-fluorouracil (5-FU) loading and subsequent tableting as a new drug formulation for slow release has been proposed using the biomineral from blue crab carapace. Due to its highly ordered 3D porous nanoarchitecture, the biogenic carbonate carrier could achieve increased effectiveness in colorectal cancer cure provided that the formulation would successfully pass through the gastric acid conditions. Following the recently proven viability of the concept by demonstrating the slow release of the drug from the carrier using the highly sensitive SERS technique, here we investigated the 5-FU release from the composite tablet drug in pH conditions replicating the gastric environment. The released drug from the tablet was studied in solutions with three relevant pH values, pH 2, pH 3, and pH 4. The 5-FU SERS spectral signature for each pH value was used to build calibration curves for quantitative SERS analysis. The results suggested a similarly slow-releasing pattern in acid pH environments to that in neutral conditions. Although biogenic calcite dissolution was expected in acid conditions, the X-ray diffraction and Raman spectroscopy showed preservation of calcite mineral along with the monohydrocalcite during acid solution exposure for two hours. The total released amount in a time course of seven hours, however, was lower in acidic pH solutions, with a maximum fraction of ~40% of the total amount of loaded drug, for pH 2, as opposed to ~80% for neutral values. Nonetheless, these results clearly prove that the novel composite drug retains its slow-releasing character in environmental conditions compatible with the gastrointestinal pH and that it is a viable and biocompatible alternative for oral delivery of anticancer drug to reach the lower gastro-intestinal tract.

Rutin bioconjugates as potential nutraceutical prodrugs: An in vitro and in ovo toxicological screening.

Rutin (RUT) is considered one the most attractive flavonoids from a therapeutic perspective due to its multispectral pharmacological activities including antiradical, anti-inflammatory, antiproliferative, and antimetastatic among others. Still, this compound presents a low bioavailability what narrows its clinical applications. To overcome this inconvenience, the current paper was focused on the synthesis, characterization, and toxicological assessment of two RUT bioconjugates obtained by enzymatic esterification with oleic acid (OA) and linoleic acid (LA)-rutin oleate (RUT-O) and rutin linoleate (RUT-L), as flavonoid precursors with improved physicochemical and biological properties. Following the enzymatic synthesis in the presence of Novozyme® 435, the two bioconjugates were obtained, their formation being confirmed by RAMAN and FT-IR spectroscopy. The in vitro and in ovo toxicological assessment of RUT bioconjugates (1-100 µM) was performed using 2D consecrated cell lines (cardiomyoblasts - H9c2(2-1), hepatocytes-HepaRG, and keratinocytes-HaCaT), 3D reconstructed human epidermis tissue (EpiDerm™), and chick chorioallantoic membranes, respectively. The results obtained were test compound, concentration-and cell-type dependent, as follows: RUT-O reduced the viability of H9c2(2-1), HepaRG, and HaCaT cells at 100 µM (to 77.53%, 83.17%, and 78.32%, respectively), and induced cell rounding and floating, as well as apoptotic-like features in the nuclei of all cell lines, whereas RUT-L exerted no signs of cytotoxicity in all cell lines in terms of cell viability, morphology, and nuclear integrity. Both RUT esters impaired the migration of HepaRG cells (at 25 µM) and lack irritative potential (at 100 µM) in vitro (tissue viability >50%) and in ovo (irritation scores of 0.70 for RUT-O, and 0.49 for RUT-L, respectively). Computational predictions revealed an increased lipophilicity, and reduced solubility, drug-likeness and drug score of RUT-O and RUT-L compared to their parent compounds-RUT, OA, and LA. In conclusion, we report a favorable toxicological profile for RUT-L, while RUT-O is dosage-limited since at high concentrations were noticed cytotoxic effects.

Comparative screening the life-time composition and crystallinity variation in gilthead seabream otoliths Sparus aurata from different marine environments.

Differences in crystallinity, structure and composition variation along the growing direction in gilthead seabream, Sparus aurata otoliths that inhabited different environments were determined to evaluate the correlation of spectroscopic and chemical data with the lifetime development and movement pattern. The Raman spectroscopy signal provided the characteristic bands whose Full Width at Half Maximum (FWHM) were used to track the signal variability. The FWHM showed an initial increase in the core area, followed by a decrease depicting two minima coinciding growth rings. The crystal discontinuity linked to annual rings was confirmed. The FWHM pattern followed cycle in the individual's activity. However, no significant correlation with FWHM and environmental factors although the slope of the FWHM variation distinguished aquaculture and costal groups from open sea and transitional, estuarine waters. Raman data were further correlated with morphological and elemental composition obtained via SEM-EDX and by LA-ICP-MS. SEM clearly confirmed CRM findings. Finally, multiparameter analysis of Ba/Ca concentrations obtained by LA-ICP-MS indicated the separation of groups associated with aquaculture and transitional waters due lowest variability in the elemental composition. Other groups are more variable possibly due to the water oligotrophic character and greater variability in prey availability in each environment. Results of the present study showed the additional potential of Raman spectroscopy as a complementary tool for inference of migration or origin of fish based on otolith composition and structure like other well-established technique.

Rapid and Application-Tailored Assessment Tool for Biogenic Powders from Crustacean Shell Waste: Fourier Transform-Infrared Spectroscopy Complemented with X-ray Diffraction, Scanning Electron Microscopy, and Nuclear Magnetic Resonance Spectroscopy.

Due to their chemical composition, richness in calcium carbonate, chitin, proteins, and pigments, and nanoporous structure, crustacean shell waste shows great potential for a wide variety of applications. Large quantities of waste shells are produced annually, meaning that they can be considered a renewable source of ecofriendly biogenic materials, which can be turned into value-added byproducts. In this paper, an IR-based technique is developed to differentiate various biogenic powders originated from crude or food-processed crustacean shells. The validity of the method is supported by cross-checking with XRD, NMR, and SEM-EDX analyses. Our goal was to determine changes in properties of waste crab shells after the two most common treatments, deproteinization and milling. We discovered that deproteinization with NaOH could be tracked from the IR absorbance intensity ratio of the υ(CH2,3) and υasym(CO3 2-) bands while milling time less influenced this ratio but induced changes in powder particle size distribution and morphology. The relative organic/inorganic ratio was different for different colored shells. Unexpectedly, waste shells stored for an average of 6 months or more were found to contain hydrated calcium carbonate (monohydrocalcite), which was absent in equivalent fresh shell composition. Deproteinization caused changes in mechanical properties of shells, making them more brittle, which resulted in a larger fraction of fine particles after powdering.

Novel Drug Carrier: 5-Fluorouracil Formulation in Nanoporous Biogenic Mg-calcite from Blue Crab Shells-Proof of Concept.

The ever-growing demand for novel, cheaper, and more effective drugs has put nanomedicine and targeted drug delivery to the forefront of scientific innovation. Owing to its porous three-dimensional (3D)-nanostructure and properties, the biogenic calcite from wasted blue crab shells is employed in the present work as a new drug carrier for 5-fluorouracil (5-FU), a drug widely used in cancer therapy. The drug solution has been loaded in the porous nanoarchitecture of the powdered biogenic material and further pelleted in tablets with a 5-FU concentration of 1.748 mg/g. Their structural and morphological properties were characterized using Raman, X-ray diffraction, and scanning electron microscopy. Confocal micro-Raman spectra of tablet surface showed a typical signal of biogenic carbonate with preserved carotenoids and carotenoproteins found in the native waste shell, while the drug Raman signal was absent, indicating its adsorption in the intricate nanoporous biogenic carrier. The slow release of the drug from the newly formulated tablet was investigated by tracking the surface-enhanced Raman scattering (SERS) signal of the tablet solution in a series of time-dependent experiments. The SERS signal quantification is achieved using the well-known SERS spectral fingerprint of 5-fluorouracil aqueous solution adsorbed on Ag nanoparticles. The proof of concept is demonstrated by quantifying the slow release of the drug through the characteristic SERS band intensity of 5-FU in a time course of 26 h. This proof of concept boosted further investigations concerning the released drug identity in simulated solutions that mimic the pH of the upper- and lower gastrointestinal tract, as well as the multiple possibilities to control porosity and composition during powdering and treatment of biogenic material, to achieve the most convenient formulation for relevant biomedical drug delivery. Nonetheless, the present results showed great promise for innovative reusing waste biogenic 3D-nanomaterials of aquatic origin as advantageous drug carriers for slow release purposes, in line with the concept of blue bioeconomy.