Antiproliferative activity and apoptosis-inducing mechanism of Amaryllidaceae alkaloid montanine on A549 and MOLT-4 human cancer cells.

The isoquinoline alkaloids found in Amaryllidaceae are attracting attention due to attributes that can be harnessed for the development of new drugs. The possible molecular mechanisms by which montanine exerts its inhibitory effects against cancer cells have not been documented. In the present study, montanine, manthine and a series of 15 semisynthetic montanine analogues originating from the parent alkaloid montanine were screened at a single test dose of 10 µM to explore their cytotoxic activities against a panel of eight cancer cell lines and one non-cancer cell line. Among montanine and its analogues, montanine and its derivatives 12 and 14 showed the highest cytostatic activity in the initial single-dose screening. However, the native montanine exhibited the greatest antiproliferative activity against cancer cells, with a lower mean IC50 value of 1.39 µM, compared to the displayed mean IC50 values of 2.08 µM for 12 and 3.57 µM for 14. Montanine exhibited the most potent antiproliferative activity with IC50 values of 1.04 µM and 1.09 µM against Jurkat and A549 cell lines, respectively. We also evaluated montanine's cytotoxicity and cell death mechanisms. Our results revealed that montanine triggered apoptosis of MOLT-4 cells via caspase activation, mitochondrial depolarisation and Annexin V/PI double staining. The Western blot results of MOLT-4 cells showed that the protein levels of phosphorylated Chk1 Ser345 were upregulated with increased montanine concentrations. Our findings provide new insights into the mechanisms underlying the cytostatic, cytotoxic and pro-apoptotic activities of montanine alkaloids in lung adenocarcinoma A549 and leukemic MOLT-4 cancer cell types.

Design of semisynthetic derivatives of the Amaryllidaceae alkaloid ambelline and exploration of their in vitro cytotoxic activities.

Ambelline, an alkaloid from the Amaryllidaceae family with a crinane-type skeleton, has not yet demonstrated any outstanding biological activity. However, its analogues prepared by derivatization of the C-11 hydroxyl group show different interesting effects. Continuing our earlier work, twelve novel aromatic esters were developed (10, 14, 16, 17, 22-25, 30-33) and studied, together with previously synthesized derivatives (2-9, 11-13, 15, 18-21, 26-29) in terms of their cytotoxic activity. The cytotoxic potential was determined on a panel of nine human cancer cell lines and one noncancerous cell line to characterize their biological activity spectrum. To describe and foresee the structure-activity relationship for further research, substances synthesized and described in our previous work were also included in this cytotoxicity study. The most significant activity was associated with analogues having methyl (10), methoxy (14-17), or ethoxy (18) substitution on the phenyl condensed to ambelline. However, the 4-chloro-3-nitrobenzoyl derivative (32) showed the most promising IC50 values, ranging from 0.6 ± 0.1 µM to 9.9 ± 0.2 µM. In vitro cytotoxicity studies indicated the most potent antiproliferative activity of 32 in a dose-dependent and time-dependent manner. Besides, 32 was found to be effective in decreasing viability and triggering apoptosis of MOLT-4 T-lymphoblastic leukemia cells.

Multimodal Contrast Agent Enabling pH Sensing Based on Organically Functionalized Gold Nanoshells with Mn-Zn Ferrite Cores.

Highly complex nanoparticles combining multimodal imaging with the sensing of physical properties in biological systems can considerably enhance biomedical research, but reports demonstrating the performance of a single nanosized probe in several imaging modalities and its sensing potential at the same time are rather scarce. Gold nanoshells with magnetic cores and complex organic functionalization may offer an efficient multimodal platform for magnetic resonance imaging (MRI), photoacoustic imaging (PAI), and fluorescence techniques combined with pH sensing by means of surface-enhanced Raman spectroscopy (SERS). In the present study, the synthesis of gold nanoshells with Mn-Zn ferrite cores is described, and their structure, composition, and fundamental properties are analyzed by powder X-ray diffraction, X-ray fluorescence spectroscopy, transmission electron microscopy, magnetic measurements, and UV-Vis spectroscopy. The gold surface is functionalized with four different model molecules, namely thioglycerol, meso-2,3-dimercaptosuccinate, 11-mercaptoundecanoate, and (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, to analyze the effect of varying charge and surface chemistry on cells in vitro. After characterization by dynamic and electrophoretic light scattering measurements, it is found that the particles do not exhibit significant cytotoxic effects, irrespective of the surface functionalization. Finally, the gold nanoshells are functionalized with a combination of 4-mercaptobenzoic acid and 7-mercapto-4-methylcoumarin, which introduces a SERS active pH sensor and a covalently attached fluorescent tag at the same time. 1H NMR relaxometry, fluorescence spectroscopy, and PAI demonstrate the multimodal potential of the suggested probe, including extraordinarily high transverse relaxivity, while the SERS study evidences a pH-dependent spectral response.

Pancracine, a Montanine-Type Amaryllidaceae Alkaloid, Inhibits Proliferation of A549 Lung Adenocarcinoma Cells and Induces Apoptotic Cell Death in MOLT-4 Leukemic Cells.

Pancracine, a montanine-type Amaryllidaceae alkaloid (AA), is one of the most potent compounds among natural isoquinolines. In previous studies, pancracine exhibited cytotoxic activity against diverse human cancer cell lines in vitro. However, further insight into the molecular mechanisms that underlie the cytotoxic effect of pancracine have not been reported and remain unknown. To fill this void, the cell proliferation and viability of cancer cells was explored using the Trypan Blue assay or by using the xCELLigence system. The impact on the cell cycle was determined by flow cytometry. Apoptosis was evaluated by Annexin V/PI and by quantifying the activity of caspases (-3/7, -8, and -9). Proteins triggering growth arrest or apoptosis were detected by Western blotting. Pancracine has strong antiproliferative activity on A549 cells, lasting up to 96 h, and antiproliferative and cytotoxic effects on MOLT-4 cells. The apoptosis-inducing activity of pancracine in MOLT-4 cells was evidenced by the significantly higher activity of caspases. This was transmitted through the upregulation of p53 phosphorylated on Ser392, p38 MAPK phosphorylated on Thr180/Tyr182, and upregulation of p27. The pancracine treatment negatively altered the proliferation of A549 cells as a consequence of an increase in G1-phase accumulation, associated with the downregulation of Rb phosphorylated on Ser807/811 and with the concomitant upregulation of p27 and downregulation of Akt phosphorylated on Thr308. This was the first study to glean a deeper mechanistic understanding of pancracine activity in vitro. Perturbation of the cell cycle and induction of apoptotic cell death were considered key mechanisms of pancracine action.

Disruption of Cell Adhesion and Cytoskeletal Networks by Thiol-Functionalized Silica-Coated Iron Oxide Nanoparticles.

One of the major obstacles that limits the use of magnetic nanoparticles in biomedical applications is their potential toxicity. In the present study, we evaluated the cytotoxic effects of thiol-functionalized silica-coated iron oxide (Fe3O4@SiO2-SH) nanoparticles using human lung epithelial cells A549. We investigated the effect of Fe3O4@SiO2-SH nanoparticles on the cell viability, proliferation, cell cycle distribution, adhesion, apoptosis, and the orientation of the cytoskeletal networks, as well as on expression of proteins involved in cell death, cell survival, and cell adhesion. We demonstrated that exposure of A549 cells to Fe3O4@SiO2-SH nanoparticles resulted in severe disruption of the actin microfilaments and microtubule cytoskeleton and reduced the size of focal adhesions. Furthermore, cell adhesion was significantly affected as well as the phosphorylation of focal adhesion kinase (FAK), extracellular-signal-regulated kinase (ERK), and p38. Our findings highlight the need for in-depth cytotoxic evaluation of nanoparticles supporting their safer use, especially in biomedical applications.

Chemical Composition of Natural Hydrolates and Their Antimicrobial Activity on Arcobacter-Like Cells in Comparison with Other Microorganisms.

Hydrolates obtained via the hydrodistillation and steam distillation of Lavandulaangustifolia Mill., Syzygiumaromaticum L., Foeniculumvulgare Mill., and Laurusnobilis L. were analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography coupled to mass spectrometry (GC-MS). Additionally, the hydrolates were evaluated for antimicrobial activity (disk-diffusion and microdilution method), influence on biofilm formation (Christensen method) and cytotoxicity of concentrated hydrolates against human cell lines (A549) by xCELLigence system. Using chemical analysis, 48, 9, 13 and 33 different components were detected in lavender, clove, fennel and laurel hydrolates, respectively. Lavender hydrolate contained the largest proportion of 1,8-cineol, linalool furanoxide, and linalool. The main components of laurel hydrolate were 1,8-cineol, 4-terpineol and α-terpineol. Fenchone and estragole were the most abundant in fennel hydrolate, and eugenol and eugenyl acetate in clove hydrolate. Concentrated hydrolates showed significant antimicrobial activity. Clove hydrolate was among the most antimicrobially active agents, most preferably against C. albicans, with an inhibition zone up to 23.5 mm. Moreover, concentrated hydrolates did not show any cytotoxic effect again8 st human A549 cells. In the presence of the non-concentrated hydrolates, significantly reduced biofilm formation was observed; however, with concentrated clove hydrolate, there was an increase in biofilm formation, e.g., of A. thereius, A. lanthieri, and A. butzleri. Research shows new findings about hydrolates that may be important in natural medicine or for preservation purposes.

Magnetic nanoparticles of Ga-substituted ε-Fe2 O3 for biomedical applications: Magnetic properties, transverse relaxivity, and effects of silica-coated particles on cytoskeletal networks.

Magnetic nanoparticles of ε-Fe1.76 Ga0.24 O3 with the volume-weighted mean size of 17 nm were prepared by thermal treatment of a mesoporous silica template impregnated with metal nitrates and were coated with silica shell of four different thicknesses in the range 6-24 nm. The bare particles exhibited higher magnetization than the undoped compound, 22.4 Am2 kg-1 at 300 K, and were characterized by blocked state with the coercivity of 1.2 T at 300 K, being thus the very opposite of superparamagnetic iron oxides. The relaxometric study of the silica-coated samples at 0.47 T revealed promising properties for MRI, specifically, transverse relaxivity of 89-168 s-1 mmol(f.u.)-1 L depending on the shell thickness was observed. We investigated the effects of the silica-coated nanoparticles on human A549 and MCF-7 cells. Cell viability, proliferation, cell cycle distribution, and the arrangement of actin cytoskeleton were assessed, as well as formation and maturation of focal adhesions. Our study revealed that high concentrations of silica-coated particles with larger shell thicknesses of 16-24 nm interfere with the actin cytoskeletal networks, inducing thus morphological changes. Consequently, the focal adhesion areas were significantly decreased, resulting in impaired cell adhesion.

Bersavine: A Novel Bisbenzylisoquinoline Alkaloidwith Cytotoxic, Antiproliferative and Apoptosis-Inducing Effects on Human Leukemic Cells.

Bersavine is the new bisbenzylisoquinoline alkaloid isolated from the Berberis vulgaris L.(Berberidaceae) plant. The results of cytotoxicity screening 48 h post-treatment showed thatbersavine considerably inhibits the proliferation and viability of leukemic (Jurkat, MOLT-4), colon(HT-29), cervix (HeLa) and breast (MCF-7) cancer cells with IC50 values ranging from 8.1 to 11 µM.The viability and proliferation of leukemic Jurkat and MOLT-4 cells were decreased after bersavinetreatment in a time- and dose-dependent manner. Bersavine manifested concentration-dependentantiproliferative activity in human lung, breast, ovarian and hepatocellular carcinoma cell linesusing a xCELLigence assay. Significantly higher percentages of MOLT-4 cells exposed to bersavineat 20 µM for 24 h were arrested in the G1 phase of the cell cycle using the flow cytometry method.The higher percentage of apoptotic cells was measured after 24 h of bersavine treatment. Theupregulation of p53 phosphorylated on Ser392 was detected during the progression of MOLT-4 cellapoptosis. Mechanistically, bersavine-induced apoptosis is an effect of increased activity ofcaspases, while reduced proliferation seems dependent on increased Chk1 Ser345 phosphorylationand decreased Rb Ser807/811 phosphorylation in human leukemic cells.

Silica coated iron oxide nanoparticles-induced cytotoxicity, genotoxicity and its underlying mechanism in human HK-2 renal proximal tubule epithelial cells.

Iron oxide nanoparticles (IONPs) have a great potential with regard to cell labelling, cell tracking, cell separation, magnetic resonance imaging, magnetic hyperthermia, targeted drug and gene delivery. However, a growing body of research has raised concerns about the possible unwanted adverse cytotoxic effects of IONPs. In the present study, the in vitro cellular uptake, antiproliferative activity, cytotoxicity, genotoxicity, prooxidant, microtubule-disrupting and apoptosis-inducing effect of Fe3O4@SiO2 and passivated Fe3O4@SiO2-NH2 nanoparticles on human renal proximal tubule epithelial cells (HK-2) have been studied. Both investigated silica coated IONPs were found to have cell growth-inhibitory activity in a time- and dose-dependent manner. Determination of cell cycle phase distribution by flow cytometry demonstrated a G1 and G2/M phase accumulation of HK-2 cells. A tetrazolium salt cytotoxicity assay at 24 h following treatment demonstrated that cell viability was reduced in a dose-dependent manner. Microscopy observations showed that both Fe3O4@SiO2 and Fe3O4@SiO2-NH2 nanoparticles accumulated in cells and appeared to have microtubule-disrupting activity. Our study also revealed that short term 1 h exposure to 25 and 100 µg/mL of silica coated IONPs causes genotoxicity. Compared with vehicle control cells, a significantly higher amount of γH2AX foci correlating with an increase in DNA double-strand breaks was observed in Fe3O4@SiO2 and Fe3O4@SiO2-NH2-treated and immunestained HK-2 cells. The investigated nanoparticles did not trigger significant ROS generation and apoptosis-mediated cell death. In conclusion, these findings provide new insights into the cytotoxicity of silica coated IONPs that may support their further safer use.

Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells.

Scoulerine is an isoquinoline alkaloid, which indicated promising suppression of cancer cells growth. However, the mode of action (MOA) remained unclear. Cytotoxic and antiproliferative properties were determined in this study. Scoulerine reduces the mitochondrial dehydrogenases activity of the evaluated leukemic cells with IC50 values ranging from 2.7 to 6.5 µM. The xCELLigence system revealed that scoulerine exerted potent antiproliferative activity in lung, ovarian and breast carcinoma cell lines. Jurkat and MOLT-4 leukemic cells treated with scoulerine were decreased in proliferation and viability. Scoulerine acted to inhibit proliferation through inducing G2 or M-phase cell cycle arrest, which correlates well with the observed breakdown of the microtubule network, increased Chk1 Ser345, Chk2 Thr68 and mitotic H3 Ser10 phosphorylation. Scoulerine was able to activate apoptosis, as determined by p53 upregulation, increase caspase activity, Annexin V and TUNEL labeling. Results highlight the potent antiproliferative and proapoptotic function of scoulerine in cancer cells caused by its ability to interfere with the microtubule elements of the cytoskeleton, checkpoint kinase signaling and p53 proteins. This is the first study of the mechanism of scoulerine at cellular and molecular level. Scoulerine is a potent antimitotic compound and that it merits further investigation as an anticancer drug.

Anticancer potential of Amaryllidaceae alkaloids evaluated by screening with a panel of human cells, real-time cellular analysis and Ehrlich tumor-bearing mice.

In this study, twenty-two Amaryllidaceae alkaloids were screened for their anticancer potential. All isolates were evaluated for antiproliferative activities on a panel of 17 human cell types of different tissue origin using WST-1 assay. In addition, we determined the antiproliferative effect with a real-time cell analysis xCELLigence system. Thereafter, to evaluate the barely known in vivo anticancer potential of the most potent molecule haemanthamine, a preliminary study was performed using an Ehrlich tumor-bearing mice model. The results showed that haemanthamine, lycorine and haemanthidine exerted the highest antiproliferative activity. The mean growth percent (GP) value after a single-dose 10 µM treatment was for haemanthamine 21%, for lycorine 21% and for haemanthidine 27% that of untreated control cells (100%). Furthermore, haemanthamine, lycorine and haemanthidine exhibited significant cytotoxicities against all the tested cell lines with individual IC50 values in the micromolar range. Dynamic real-time measures of impedance by xCELLigence indicated that these three compounds suppress cell proliferation after 10 h of treatment at a concentration of 10 µM or higher. Regrettably, in a follow-up in vivo antitumor activity study, haemanthamine showed no statistically significant reduction in the tumor size with no prolongation of survival time of Ehrlich tumor-bearing mice. Taken together, these results provide a new clue and guidance for exploiting Amaryllidaceae alkaloids as anticancer agents.

Synthesis, characterization and in vitro evaluation of substituted N-(2-phenylcyclopropyl)carbamates as acetyl- and butyrylcholinesterase inhibitors.

A serie of O-substituted N-2-phenylcyclopropylcarbamates was prepared and characterized. These carbamates were tested as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). It was found, that these compounds exhibit moderate inhibition activity with values of IC50 in the range of 54.8-94.4 µM (for AChE) and up to 5.8 µM (for BChE). The AChE/BChE selectivity for each carbamate was calculated. These values varied from 0.50 to 9.46, two carbamate derivatives inhibited only AChE selectively. The most promising derivative was prepared in all optically pure forms (four isomers). It was found that individual stereoisomers differed only slightly in the inhibition ability. The cytotoxicity of all carbamates was evaluated using the standard in vitro test with Jurkat cells. With regard to their inhibition activity and cytotoxicity as well as easy preparation, O-substituted N-2-phenylcyclopropylcarbamates can be considered as promising compounds for potential medicinal applications.

Comparative cytotoxicity of chelidonine and homochelidonine, the dimethoxy analogues isolated from Chelidonium majus L. (Papaveraceae), against human leukemic and lung carcinoma cells.

BACKGROUND: The search for new anticancer compounds is a crucial element of natural products research. PURPOSE: In this study the effects of naturally occurring homochelidonine in comparison to chelidonine on cell cycle progression and cell death in leukemic T-cells with different p53 status are described. METHODS: The mechanism of cytotoxic, antiproliferative, apoptosis-inducing effects and the effect on expressions of cell cycle regulatory proteins was investigated using XTT assay, Trypan blue exclusion assay, flow cytometry, Western blot analysis, xCELLigence, epi-fluorescence and 3D super resolution microscopy. A549 cells were used for xCELLigence, clonogenic assay and for monitoring microtubule stability. RESULTS: We found that homochelidonine and chelidonine displayed significant cytotoxicity in examined blood cancer cells with the exception of HEL 92.1.7 and U-937 exposed to homochelidonine. Unexpectedly, homochelidonine and chelidonine-induced cytotoxicity was more pronounced in Jurkat cells contrary to MOLT-4 cells. Homochelidonine showed an antiproliferative effect on A549 cells but it was less effective compared to chelidonine. Biphasic dose-depended G1 and G2/M cell cycle arrest along with the population of sub-G1 was found after treatment with homochelidonine in MOLT-4 cells. In variance thereto, an increase in G2/M cells was detected after treatment with homochelidonine in Jurkat cells. Treatment with chelidonine induced cell cycle arrest in the G2/M cell cycle in both MOLT-4 and Jurkat cells. MOLT-4 and Jurkat cells treated with homochelidonine and chelidonine showed features of apoptosis such as phosphatidylserine exposure, a loss of mitochondrial membrane potential and an increase in the caspases -3/7, -8 and -9. Western blots indicate that homochelidonine and chelidonine exposure activates Chk1 and Chk2. Studies conducted with fluorescence microscopy demonstrated that chelidonine and homochelidonine inhibit tubulin polymerization in A549 cells. CONCLUSION: Collectively, the data indicate that chelidonine and homochelidonine are potent inducers of cell death in cancer cell lines, highlighting their potential relevance in leukemic cells.

Synthesis, structural characterization, docking, lipophilicity and cytotoxicity of 1-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl carbamates, novel acetylcholinesterase and butyrylcholinesterase pseudo-irreversible inhibitors.

In the current study, sixteen novel derivatives of (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine were synthesized as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. Chemical structures together with purity of the synthesized compounds were substantiated by IR, (1)H, (13)C, (19)F NMR, high resolution mass spectrometry and elemental analysis. The optical activities were confirmed by optical rotation measurements. The synthesized compounds were evaluated for their AChE and BChE inhibitory activities. In addition, the cytotoxicity of the most active compounds was investigated against human cell lines employing XTT tetrazolium salt reduction assay and xCELLigence system allowing a label-free assessment of the cells proliferation. Our results demonstrated that the inhibitory mechanism was confirmed to be pseudo-irreversible, in line with previous studies on carbamates. Compounds indicated as 3b, 3d, 3l and 3n showed the best AChE inhibitory activity of all the evaluated compounds and were up to tenfold more potent than standard drug rivastigmine. The binding mode was determined using state-of-the-art covalent docking and scoring methodology. The obtained data clearly demonstrated that 3b, 3d, 3l and 3n benzothiazole carbamates possess high inhibitory activity against AChE and BChE and concurrently negligible cytotoxicity. In conclusion, our results indicate, that these derivatives could be promising in an effective therapeutic intervention for Alzheimer's disease.

Specific inhibition of Wee1 kinase and Rad51 recombinase: a strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks.

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.

The effect of Amaryllidaceae alkaloids haemanthamine and haemanthidine on cell cycle progression and apoptosis in p53-negative human leukemic Jurkat cells.

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line.

Synthesis and characterization of a pH-sensitive conjugate of isoniazid with Fe3O4@SiO2 magnetic nanoparticles.

The Letter describes the preparation and characterization of a conjugate of isoniazid (INH) with magnetic nanoparticles Fe3O4@SiO2 115±60 nm in size. The INH molecules were attached to the surface of nanoparticles by a covalent pH-sensitive amidine bond. The conjugate was characterized by X-ray diffraction, SEM, dynamic light scattering, IR spectroscopy and microanalysis. The conjugate released isoniazid under in vitro conditions (pH=4; 37 °C; t1/2≈115 s). In addition, the cytotoxicity of the Fe3O4@SiO2-INH conjugate was evaluated in SK-BR-3 cells using the xCELLigence system.

Synthesis and in vitro evaluation of new derivatives of 2-substituted-6-fluorobenzo[d]thiazoles as cholinesterase inhibitors.

A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthesised and characterised by IR, (1)H, (13)C and (19)F NMR spectroscopy and HRMS. Purity was checked by elemental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure-activity relationships reflecting the dependence of cholinesterase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinesterase was determined using molecular docking of the most active compound.

Albumin-coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies: application to the capture of epithelial cancer cells.

Monodisperse (4 µm) macroporous crosslinked poly(glycidyl methacrylate) (PGMA) microspheres for use in microfluidic immunomagnetic cell sorting, with a specific application to the capture of circulating tumor cells (CTCs), were prepared by multistep swelling polymerization in the presence of cyclohexyl acetate porogen and hydrolyzed and ammonolyzed. Iron oxide was then precipitated in the microspheres to render them magnetic. Repeated precipitation made possible to raise the iron oxide content to more than 30 wt %. To minimize nonspecific adsorption of the microspheres in a microchannel and of cells on the microspheres, they were coated with albumin crosslinked with glutaraldehyde. Antibodies of epithelial cell adhesion molecule (anti-EpCAM) were then immobilized on the albumin-coated magnetic microspheres using the carbodiimide method. Capture of breast cancer MCF7 cells as a model of CTCs by the microspheres with immobilized anti-EpCAM IgG was performed in a batch experiment. Finally, MCF7 cells were captured by the anti-EpCAM-immobilized albumin-coated magnetic microspheres in an Ephesia chip. A very good rejection of lymphocytes was achieved. Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized anti-EpCAM seem to be promising for capture of CTCs in a microfluidic device.

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae, Anura).

Using histological techniques and computer-aided three-dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the "ventromedial diverticulum") is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes.