A New Paclitaxel Formulation Based on Secretome Isolated from Mesenchymal Stem Cells Shows a Significant Cytotoxic Effect on Osteosarcoma Cell Lines.

BACKGROUND: Osteosarcoma (OS) represents a rare cancer with an unfavorable prognosis that needs innovative treatment. The aim was to isolate a secretome from mesenchymal stem cells (MSCs) that are treated with paclitaxel (PTX)-containing microvesicles as a drug delivery system and analyze its cytotoxic effects on OS cell lines (SJSA, MG63, and HOS). METHODS: Three batches of secretome (SECR-1, SECR-2, and SECR-3) were produced from three bone marrow (BM) MSCs samples treated for 24 h with 15 µg/mL of PTX or with a standard medium. The viability of the OS cell lines after 5 days of exposure to SECR-1-2-3 (pure and diluted to 1:2 and 1:4) was analyzed with an MTT assay. The same SECR batches were analyzed with high-performance liquid chromatography (HPLC) and with a nanoparticle tracking assay (NTA). RESULTS: A statistically significant decrease in the viability of all OS cell lines was observed after treatment with SECR-PTX 1-2-3 in a dose-response manner. The NTA analyses showed the presence of nanoparticles (NPs) with a mean size comparable to that of extracellular vesicles (EVs). The HPLC analyses detected the presence of PTX in minimal doses in all SECR batches. CONCLUSIONS: This proof-of-concept study showed that the conditioned medium isolated from MSCs loaded with PTX had a strong cytotoxic effect on OS cell lines, due to the presence of EV and PTX.

The challenge of o-phenylphenol detection in coffee: How "OPP-conjugates" hide their presence in green and roasted samples.

o-Phenylphenol (OPP) is not a commonly used pesticide in the coffee production chain. Although it has only been detected in roasted coffee, it is unlikely that OPP can be formed during roasting. Its acidic nature may lead to the formation of conjugates with natural matrix components. The objective of this study is to optimize an analytical method to discover how these conjugates may mask the presence of OPP in coffee. Sample extraction with hexane followed by basic hydrolysis and then a QuEChERS method allows the presence of OPP to be quantitatively detected via UPLC-MS/MS. The optimized method was applied to the same Arabica coffee (Brazil), and the quantification of comparable amounts of OPP was observed in both green and roasted samples (34.8 vs 32.2 µg/kg). The optimized procedure detected twice the amount of OPP in roasted samples, compared to the QuEChERS method, suggesting that roasting causes the partial hydrolysis of OPP conjugates.

Lack of interaction of the fluorosurfactant C6O4 with human renal transporters: In vitro/in silico analysis.

C6O4 is a water soluble perfluoroether carboxylic acid ammonium salt used as surfactant in the synthesis of fluoropolymers. Available experimental data in rats exposed by the oral route indicate it is eliminated in urine. Previous studies with various linear perfluorocarboxylic acids have suggested that these compounds are substrates of renal membrane transporters in rats and humans, and that the interaction with basal and apical membrane transporters can influence the elimination kinetic by these organisms and explain, in part, the observed differences in the respective half-lives. In particular, apical transporters may contribute to the reuptake of these exogenous compounds from the tubule lumen. The present study was designed to investigate the uptake of C6O4 in two renal cell lines transiently transfected with the human apical membrane transporters, organic anion transporter 4 (OAT4), and urate transporter 1 (URAT1). The uptake of the linear perfluorohexanoic acid (PFC6) was evaluated in parallel. While the uptake of the conjugated steroid estrone-3-sulfate (E3S), a known substrate for renal transporters, and of PFC6 was clearly observed in both cell types transfected with either OAT4 or URAT1, no significant uptake of C6O4 was measured under the same test conditions. The results of the transporter's functionality measured in vitro were consistent with molecular docking simulations. Both outward and inward models of the transporters showed a reduced interaction between C6O4 and URAT1 or OAT4. In contrast, more stable interactions were predicted for PFC6 and PFOA, as well as for the E3S substrate, as shown by the respective docking scores reflecting the binding strength and by the poses assumed in the transporter channels. Altogether, the in vitro and in silico modeling results showed a low reuptake potential and limited interactions of C6O4 molecule with two human apical membrane transporters, contrasting with the more efficient reuptake of PFC6 from the tubule lumen. These results suggest reabsorption from the proximal tubule by apical renal transporters is not likely to interfere with the elimination pathway of C6O4 in humans.

Determination of trace antibiotics in water and milk via preconcentration and cleanup using activated carbons.

CPAC-SPE-HPLC (coconut powdered activated carbon -SPE- HPLC) has been developed for the determination of antibiotic (ABX), sulfamonomethoxine sodium (SMM), oxytetracycline (OTC), ceftiofur hydrochloride (CEF) and marbofloxacin (MAR), in water and milk. Over 99.0% SMM and OTC were recovered from 20 mL of 0.5 µg/mL ABX solution using 10 mg-CPAC for adsorption and 2 mL of 30% NH4OH/EtOH (1/19 v/v) for elution. Similarly, over 99.0% CEF and MAR were recovered using 15 mg-CPAC and 2 mL of 30% NH4OH/n-PrOH (1/19 v/v). Moreover, the recovery efficiencies of various ABX from 5 to 80 mL of 0.02-2.00 µg/mL medicated milk containing 10 mM EDTA are ordered as follows: OTC (99.3%), SMM (99.1%) > CEF (68.9%) > MAR (61.4%). No interference towards HPLC analysis were observed with elution using 2 mL of 30% NH4OH/EtOH (1/19 v/v). Furthermore, much lower limit of detections (0.02 µg/mL) than the maximum residual limits from European Commission (0.075-0.100 µg/mL) were obtained.

Microwave-Assisted, One-Pot Synthesis of Doxycycline under Heterogeneous Catalysis in Water.

The selective synthesis of active pharmaceutical molecules is a challenging issue, particularly when attempting to make the reactions even more sustainable. The present work focuses on the microwave-assisted hydrogenolysis of oxytetracycline to selectively produce α-doxycycline. Although the combination of microwave irradiation and a heterogeneous rhodium catalyst provided good conversions, the selective synthesis of active α-doxycycline was only achieved when an oxytetracycline-cyclodextrin complex was used as the starting material, giving the desired product at 34.0% yield in a one-step reaction under very mild conditions.

Effects of Vanadyl Complexes with Acetylacetonate Derivatives on Non-Tumor and Tumor Cell Lines.

Vanadium has a good therapeutic potential, as several biological effects, but few side effects, have been demonstrated. Evidence suggests that vanadium compounds could represent a new class of non-platinum, metal antitumor agents. In the present study, we aimed to characterize the antiproliferative activities of fluorescent vanadyl complexes with acetylacetonate derivates bearing asymmetric substitutions on the ß-dicarbonyl moiety on different cell lines. The effects of fluorescent vanadyl complexes on proliferation and cell cycle modulation in different cell lines were detected by ATP content using the CellTiter-Glo Luminescent Assay and flow cytometry, respectively. Western blotting was performed to assess the modulation of mitogen-activated protein kinases (MAPKs) and relevant proteins. Confocal microscopy revealed that complexes were mainly localized in the cytoplasm, with a diffuse distribution, as in podocyte or a more aggregate conformation, as in the other cell lines. The effects of complexes on cell cycle were studied by cytofluorimetry and Western blot analysis, suggesting that the inhibition of proliferation could be correlated with a block in the G2/M phase of cell cycle and an increase in cdc2 phosphorylation. Complexes modulated mitogen-activated protein kinases (MAPKs) activation in a cell-dependent manner, but MAPK modulation can only partly explain the antiproliferative activity of these complexes. All together our results demonstrate that antiproliferative effects mediated by these compounds are cell type-dependent and involve the cdc2 and MAPKs pathway.

Effects of the Molecular Weight of Hyaluronic Acid in a Carbon Nanotube Drug Delivery Conjugate.

Hyaluronic acid (HA) is a ubiquitous biopolymer involved in many pathophysiological roles. One HA receptor, the cluster of differentiation CD44 protein, is often overexpressed in tumor cells. As such, HA has attracted considerable interest in the development of drug delivery formulations, given its intrinsic targetability toward CD44 overexpressing cells. The present study is focused on examining the correlation of HA molecular weight with its targetability properties. A library of conjugates obtained by linking the amino group of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) to the carboxylic residues of HA of different molecular weight (6.4, 17, 51, 200, and 1,500 kDa) were synthesized and fully characterized. The HA-DMPE conjugates were then used to non-covalently functionalize the highly hydrophobic single-walled carbon nanotubes (CNT), and further encapsulate the anticancer drug doxorubicin (DOX). Our results show that the complexes DOX/CNT/HA-DMPE maintain very good and stable dispersibility. Drug release studies indicated a pH-responsive release of the drug from the nanocarrier. Cell viability tests demonstrated that all HA modified CNTs have good biocompatibility, and specific targeting toward cells overexpressing the CD44 receptor. Among all the molecular weights tested, the 200 kDa HA showed the highest increase in cellular uptake and cytotoxic activity. All these promising attributes make CNT/HA200-DMPE a "smart" platform for tumor-targeted delivery of anticancer agents.

Exploiting Lipid and Polymer Nanocarriers to Improve the Anticancer Sonodynamic Activity of Chlorophyll.

Sonodynamic therapy is an emerging approach that uses low-intensity ultrasound to activate a sonosensitizer agent triggering its cytotoxicity for selective cancer cell killing. Several molecules have been proposed as sonosensitizer agents, but most of these, as chlorophyll, are strongly hydrophobic with a low selectivity towards cancer tissues. Nanocarriers can help to deliver more efficiently the sonosensitizer agents in the target tumor site, increasing at the same time their sonodynamic effect, since nanosystems act as cavitation nuclei. Herein, we propose the incorporation of unmodified plant-extracted chlorophyll into nanocarriers with different composition and structure (i.e., liposomes, solid lipid nanoparticles and poly(lactic-co-glycolic acid) nanoparticles) to obtain aqueous formulations of this natural pigment. The nanocarriers have been deeply characterized and then incubated with human prostatic cancer cells (PC-3) and spheroids (DU-145) to assess the influence of the different formulations on the chlorophyll sonodynamic effect. The highest sonodynamic cytotoxicity was obtained with chlorophyll loaded into poly(lactic-co-glycolic acid) nanoparticles, showing promising results for future clinical investigations on sonodynamic therapy.

A Cross-Flow Ultrasound-Assisted Extraction of Curcuminoids from Curcuma longa L.: Process Design to Avoid Degradation.

Rhizomes of Curcuma longa L. are well known for their content of curcuminoids, which are compounds with interesting biological activity against various inflammatory states and diseases. Curcuminoids can degrade during processing. This piece of work investigates fast, efficient and cost-effective metabolite recovery from turmeric under ultrasound-assisted extraction (UAE). An analytical evaluation of curcuminoid stability under sonication in different solvents is reported for the first time. HPLC and quantitative 1H-NMR were used. Under the applied conditions, EtOAc was found to be the optimal extraction medium, rather than EtOH, due to its lower radical generation, which facilitates better curcuminoid stability. Kinetic characterization, by means of the Peleg equation, was applied for single-step UAE on two different rhizome granulometries. Over a time of 90 min, maximum extraction yields were 25.63% and 47.56% for 6 and 2 mm matrix powders, respectively. However, it was observed that the largest portion of curcuminoid recovery was achieved in the first 30 min. Model outcomes were used as the basis for the design of a suitable multi-step cross-flow approach that supports and emphasizes the disruptive role of cavitation. The maximum curcuminoid yield was achieved over three steps (92.10%) and four steps (80.04%), for lower and higher granulometries, respectively. Finally, the central role of the solvent was further confirmed by turmeric oleoresin purification. The EtOAc extract was purified via crystallization, and a 95% pure curcuminoid product was isolated without any chromatographic procedure. No suitable crystallization was observed for the EtOH extract.

Laser-Synthesis of NV-Centers-Enriched Nanodiamonds: Effect of Different Nitrogen Sources.

Due to the large number of possible applications in quantum technology fields-especially regarding quantum sensing-of nitrogen-vacancy (NV) centers in nanodiamonds (NDs), research on a cheap, scalable and effective NDs synthesis technique has acquired an increasing interest. Standard production methods, such as detonation and grinding, require multistep post-synthesis processes and do not allow precise control in the size and fluorescence intensity of NDs. For this reason, a different approach consisting of pulsed laser ablation of carbon precursors has recently been proposed. In this work, we demonstrate the synthesis of NV-fluorescent NDs through pulsed laser ablation of an N-doped graphite target. The obtained NDs are fully characterized in the morphological and optical properties, in particular with optically detected magnetic resonance spectroscopy to unequivocally prove the NV origin of the NDs photoluminescence. Moreover, to compare the different fluorescent NDs laser-ablation-based synthesis techniques recently developed, we report an analysis of the effect of the medium in which laser ablation of graphite is performed. Along with it, thermodynamic aspects of the physical processes occurring during laser irradiation are analyzed. Finally, we show that the use of properly N-doped graphite as a target for laser ablation can lead to precise control in the number of NV centers in the produced NDs.

A community-built calibration system: The case study of quantification of metabolites in grape juice by qNMR spectroscopy.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

SWCNT-porphyrin nano-hybrids selectively activated by ultrasound: an interesting model for sonodynamic applications.

Sonodynamic therapy (SDT) is an innovative anticancer approach, based on the excitation of a given molecule (usually a porphyrin) by inertial acoustic cavitation that leads to cell death via the production of reactive oxygen species (ROS). This study aims to prepare and characterize nanosystems based on porphyrin grafted carbon nanotubes (SWCNTs), to understand some aspects of the mechanisms behind the SDT phenomenon. Three different porphyrins have been covalently linked to SWCNTs using either Diels-Alder or 1,3-dipolar cycloadditions. ROS production and cell viability have been evaluated upon ultrasound irradiation. Despite the low porphyrin content linked on the SWCNT, these systems have shown high ROS production and high tumour-cell-killing ability. The existence of a PET (photoinduced electron transfer)-like process would appear to be able to explain these observations. Moreover, the demonstrated ability to absorb light limits the impact of side effects due to light-excitation.

Microwave Irradiation in Micro- Meso-Fluidic Systems; Hybrid Technology has Issued the Challenge.

A combination of microwave irradiation and flow chemistry has been described as a promising smart and hyphenated technology that can fuse and synergize the benefits of the techniques. The cells and tissues of all living organisms promote a huge number of bioorganic reactions that occur as flow systems and not the batch-type conditions typically used by chemists and biotechnologists. Microwave-assisted chemical conversion carried out in continuous flow mode with micro- or meso-channel reactors can offer significant processing advantages, including improved thermal exchange, energy efficiency, safety, mixing control, a wider range of reaction conditions, repeatability and scalability as well as dramatic reductions in side-reactions and degradations. This review will discuss relevant examples of organic synthesis and nanoparticles production performed in continuous flow mode with integrated microwave irradiation in micro- or mesofluidic systems.

Stearoyl-Chitosan Coated Nanoparticles Obtained by Microemulsion Cold Dilution Technique.

Chitosan is an excipient which has been studied thoroughly in research works thanks to its positive characteristics such as muco-adhesiveness and ability to open epithelial-tight-junctions. In this article, lipophilic stearoyl chitosan (ST-CS) was synthetized in order to anchor this polymer to lipid nanoparticles and prepare ST-CS-coated nanoparticles (ST-CS-NP) using the microemulsion cold dilution technique. Curcumin (CURC) was used as model drug. CURC-ST-CS-NP were characterized by dimensional analysis, zeta potential, drug entrapment, drug release; tested in vitro on Human Umbilical Vein Endothelial Cell (HUVEC) cells to study its cytotoxicity and on human pancreatic cancer cells (PANC-1) to determine inhibition ability; tested in rats to determine CURC blood profiles and biodistribution. CURC-ST-CS-NP had mean diameters in the range 200⁻400 nm and CURC entrapment up to 73%. These systems did not show cytotoxicity on HUVEC cells at all tested dilutions and revealed to be more effective than free CURC solution on PANC-1 cells at 5 and 10 µM CURC. Blood profile studies evidenced as CURC entrapment in NP prolonged the permanence of drug in the systemic circulation compared to CURC solution due to a certain stealth property of NP, probably attributable to hydrophilic chitosan coating. Biodistribution studies showed a smaller CURC concentration in RES organs when CURC-ST-CS-NP were administered.

N-Acetyl-3-aminopyrazoles block the non-canonical NF-kB cascade by selectively inhibiting NIK.

NF-κB-inducing kinase (NIK), an oncogenic drug target that is associated with various cancers, is a central signalling component of the non-canonical pathway. A blind screening process, which established that amino pyrazole related scaffolds have an effect on IKKbeta, led to a hit-to-lead optimization process that identified the aminopyrazole 3a as a low µM selective NIK inhibitor. Compound 3a effectively inhibited the NIK-dependent activation of the NF-κB pathway in tumour cells, confirming its selective inhibitory profile.

A novel synthesis of N-hydroxy-3-aroylindoles and 3-aroylindoles.

A straightforward indole synthesis via annulation of C-nitrosoaromatics with conjugated terminal alkynones was realised achieving a simple, highly regioselective, atom- and step economical access to 3-aroylindoles in moderate to good yields. Further functionalizations of indole scaffolds were investigated and an easy way to JWH-018, a synthetic cannabinoid, was achieved.

Lipophilic Prodrug of Floxuridine Loaded into Solid Lipid Nanoparticles: In Vitro Cytotoxicity Studies on Different Human Cancer Cell Lines.

Floxuridine is a very effective drug with high potency in the treatment of various tumors but its utility is limited by its low efficiency of cellular uptake. In order to improve the floxuridine efficiency of cellular uptake, lipophilic prodrug of floxuridine (3',5'-distearoyl-5-fluoro-2'-deoxyuridine) was synthetized and loaded into behenic acid nanoparticles produced by fatty acid coacervation technique. Generally, spherical shaped SLN with mean diameters below 300 nm were obtained. Distearoyl-floxuridine was loaded in SLN with high entrapment efficiency (from 70.8 to 82.8%). In Vitro cytotoxicity studies on different human cancer cell lines (M14, HT-29 and MDA-MB231) were performed in order to test the ability of distearoyl-floxuridine-SLN to inhibit the cancer cell growth. In MTT test distearoyl floxuridine SLN showed a greater efficacy than floxuridine on all cancer cell lines revealing an efficiency about 100 times higher. Also clonogenic assay showed a higher cytotoxicity of distearoyl-floxuridine-SLN compared to floxuridine but the difference between the formulations was only about 10 times. In conclusion, SLN proved to be a promising vehicle to increase the floxuridine efficacy in cancer therapy.

Porphyrin-Loaded Pluronic Nanobubbles: A New US-Activated Agent for Future Theranostic Applications.

Sonodynamic therapy (SDT) has become a promising noninvasive approach for cancer therapy. The treatment exploits the ability of particular molecules (i.e., porphyrins) to be excited by ultrasound and produce reactive oxygen species (ROS) during their decay process. These reactive species, in turn, result in cell death. To capitalize on the real-time visualization and on-demand delivery of ultrasound contrast agents, this study aims to combine porphyrins with nanobubbles (NBs) to obtain an ultrasound-activated theranostic agent that exploits the SDT activity in vitro. Two porphyrin classes, exposing different hydrophobic side chains, were synthesized. NB size and encapsulation efficiency were markedly dependent on the porphyrin structure. The combination of these porphyrin and NBs resulted in a significant reduction in cell viability upon sonication in pilot studies performed on the LS 174T colorectal cancer cell line.

EPR and photophysical characterization of six bioactive oxidovanadium(IV) complexes in the conditions of in vitro cell tests.

A number of oxidovanadium(IV) complexes have been reported to display anticancer activity. A theranostic approach, based on the simultaneous observation of both the effect of oxidovanadium(IV) complexes on cell viability and the disclosure of their intracellular fate, is possible by using oxidovanadium(IV) complexes functionalized with fluorescent ligands. In the present study we accomplished the characterization of six oxidovanadium(IV) complexes in conditions close to those employed for in vitro administration. In particular, we investigated the light harvesting properties of such complexes in the presence of a dimethylsulphoxide/aqueous buffer mixture, and we found that one complex exhibits a quantum yield suitable for confocal microscopy investigations. EPR investigations in the same conditions provide information about the presence of ligands' substitution processes. Finally, the electrochemical properties of all complexes were determined by cyclic voltammetry. The overall results show that these complexes exhibit an average stability in solution; EPR data confirm that DMSO enter the first coordination sphere of oxidovanadium(IV) and suggest the occurrence of partial ligand substitution in the dimethylsulphoxide/aqueous buffer mixture.

4-Hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide: a novel inhibitor of the canonical NF-κB cascade.

The NF-κB signaling pathway is a validated oncological target. Here, we applied scaffold hopping to IMD-0354, a presumed IKKß inhibitor, and identified 4-hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide (4) as a nM-inhibitor of the NF-κB pathway. However, both 4 and IMD-0354, being potent inhibitors of the canonical NF-κB pathway, were found to be inactive in human IKKß enzyme assays.