Microfluidics Fabrication of Monodisperse Biocompatible Phospholipid Vesicles for Encapsulation and Delivery of Hydrophilic Drug or Active Compound

We encapsulate the hydrophilic anti-cancer drug doxurubicin hydrochloride (DOX) with about 94% drug encapsulation efficiency, either alone or with nanomagnetite, in monodisperse biocompatible phospholipid vesicles. Glass capillary microfluidics is used to generate monodisperse water in oil in water (w/o/w) double-emulsion templates with a core–shell structure by using a mixture of liquid unsaturated phospholipids and powdered saturated phospholipid. This combination would overcome the low transition temperature of unsaturated powdered phospholipid and the solubility limitation of saturated phospholipid, as well as improving the fabrication of stable monodisperse phospholipid vesicles. The double-emulsion droplet is controlled from 50 to 200 μm according to different flow rates, and the final phospholipid vesicles are retained after a solvent removal step by dewetting. DOX-loaded phospholipid vesicles show sustained release compared with free DOX water solution. The in vitro cell viability of 100 μg/mL phospholipid vesicles on HeLa or MCF-7 cells after 24 h incubation at 310 K is above 90%, confirming the excellent biocompatibility of the phospholipid vesicles. These biocompatible phospholipid vesicles are promising oral drug delivery vehicles for biomedical applications and magnetic resonance imaging contrast agents for biomedical diagnosis

c-FLIP and the NOXA/Mcl-1 axis participate in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells

<div><p>Choroidal melanoma is the most common primary malignant intraocular tumor, and very few effective therapies are available to treat it. Our study aimed to understand whether pemetrexed plus cisplatin exerts a beneficial synergistic effect in human choroidal melanoma cells and to delineate the underlying molecular mechanism. To accomplish these aims, we treated choroidal melanoma cells with pemetrexed and cisplatin and assessed cell survival with SRB and MTT assays. Proteins were detected using western blotting analysis. NOXA and CHOP were knocked down with siRNA. We found that pemetrexed or cisplatin alone inhibited survival and induced apoptosis in human choroidal melanoma cells. Furthermore, the expression levels of c-FLIP, an anti-apoptotic protein in the extrinsic apoptosis pathway, and Mcl-1, an anti-apoptotic protein in the intrinsic apoptosis pathway, were decreased by pemetrexed or cisplatin respectively, while the expression of a pro-apoptotic protein in the intrinsic apoptosis pathway, NOXA, was up-regulated. Moreover, pemetrexed or cisplatin alone increased the protein expression of the endoplasmic reticulum stress markers IRE1α, Bip and CHOP. Silencing CHOP expression reduced NOXA expression. These findings suggest that the pemetrexed or cisplatin induced intrinsic apoptosis via activation of the ER stress response. Importantly, combining the two compounds more strongly induced apoptosis. Following the cotreatment, CHOP and NOXA expression increased, while c-FLIP and Mcl-1 expression decreased, and these effects were more pronounced than when using either compound alone. This result suggests that pemetrexed and cisplatin synergistically activate ER stress response-induced apoptosis in choroidal melanoma cells. To summarize, the c-FLIP and NOXA/Mcl-1 axis participated in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells. Intrinsic apoptosis was induced via activation of the ER stress response. Our study provides important mechanistic insights into potential cancer treatment with pemetrexed plus cisplatin and enriches our understanding of human choroidal melanoma.</p></div

Low-dose pemetrexed plus cisplatin has a synergistic effect in reducing c-FLIP expression in choroidal melanoma cells.

<p>OCM1 and M619 cells were treated with 1.25μM pemetrexed combined with increasing doses of cisplatin respectively for 24 hours and then harvested for western blotting analysis. The expression of c-FLIP was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. All data are presented as the mean ± S.D.</p

Low-dose pemetrexed plus cisplatin synergistically up-regulates NOXA expression and down-regulates Mcl-1 expression in choroidal melanoma cells.

<p>(A) To analyze the synergistic effect of the co-treatment, the indicated cells were treated with the indicated concentrations of the chemotherapeutic drugs for 24 hours and then harvested for western blotting. NOXA and Mcl-1 expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. OCM1 and M619 cells were seeded in 6-well plates and transfected with control or NOXA siRNA on the second day. (B, C, D)At 48 hours after transfection, the cells were treated with 1.25 μmol/L pemetrexed combined with 5 μmol/L cisplatin for another 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. All data are presented as the mean ± S.D.</p

Pemetrexed and cisplatin inhibit cell growth and induce apoptosis in a concentration-dependent and time-dependent manner.

<p>(A, D) OCM1 and M619 cells were seeded in 96-well plates and treated with increasing doses of pemetrexed or cisplatin for 36 hours. Cell survival was estimated using an SRB assay. The expression of proteins related to apoptosis in the pemetrexed or cisplatin group was examined by western blotting. (B, E) To perform a dose-gradient assay, cells were treated with increasing doses of pemetrexed or cisplatin for 36 hours. (C, F) To perform a time-gradient assay, cells were treated with 2.5 μmol/L pemetrexed or 5 μmol/L cisplatin for the indicated time, then, western blotting analysis was used to determine the expression levels of apoptosis-related proteins. CF: cleaved form. CFs: plural of cleaved form. All data are presented as the mean ± S.D.</p

Low-dose pemetrexed plus cisplatin synergistically work to inhibit survival and induce apoptosis in human choroidal melanoma cells.

<p>(A) OCM1 and (B) M619 cells were seeded in 96-well plates and treated with increasing doses of pemetrexed combined with various doses of cisplatin respectively for 24 hours. The IC50 values for the individual components and the drug combination were calculated using GraphPad Prism 5.0 software. The IC50 values for pemetrexed and cisplatin were 21.04μM and 37.87μM respectively while the values were 4.847μM, 2.95μM and 1.77μM for various concentrations of drug combination respectively in OCM1 cells. Meanwhile, the IC50 values for pemetrexed and cisplatin were 46.38μM and 56.62μM respectively and the values were 2.911μM, 1.512μM and 0.8627μM for various concentrations of drug combination respectively in M619 cells. (C, D) The synergistic effect of pemetrexed and cisplatin was analyzed using isobolograms. X-axes represented for cisplatin concentration while y-axes represented for pemetrexed concentration. An isobole was constructed by joining the points that represented the IC50 values of pemetrexed and cisplatin, and the dots underlying the isobole represented the IC50 values of the combination treatment. (E, F, G) OCM1 and M619 cells were treated with various concentrations of the chemotherapeutic drugs for 24 hours and then harvested for western blotting and apoptosis analysis. CF: cleaved form. CFs: plural of cleaved form. All data are presented as the mean ± S.D.</p

ER stress-related proteins are clearly up-regulated following treatment with pemetrexed in combination with cisplatin.

<p>To analyze the synergistic effects of the co-treatment, the indicated cells were treated with the indicated concentrations of chemotherapeutic drugs for 24 hours and then harvested for western blotting analysis. IRE1α and Bip expression was quantified using Image J software and analyzed with GraphPad Prism 5.0 software. All data are presented as the mean ± S.D.</p

Sex-specific correlation between emotion regulation ability and regional gray matter volume.

<p>A: The right DLPFC, where the interaction between sex and emotion regulation ability was found, is rendered in the e Montreal Neurological Institute (MNI) space. B: The anatomical cluster that extends from the left brainstem to the left hippocampus, the left amygdala and the insular cortex, where the interaction between sex and emotion regulation ability was found, is rendered in the MNI space. C: Scatter plots depicting correlations between rGMV of the right DLPFC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097071#pone.0097071-Killgore1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097071#pone.0097071-Koechlin1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097071#pone.0097071-Ortigue1" target="_blank">[60]</a> and individual variability in emotion regulation ability in males (N = 133, r = 0.30, p<0.001) and females (N = 159, r =  −0.08, p>0.05). D: Scatter plots depicting correlations between rGMV of the anatomical cluster that extends from the left brainstem to the left hippocampus, the left amygdala and the insular cortex [0, −14, −10] and individual variability in emotion regulation ability in males (N = 133, r = 0.20, p<0.05) and females (N = 159, r =  −0.11, p>0.05).</p

Regions correlating with emotion regulation ability.

<p><i>Note</i>: MNI  =  Montreal Neurological Institute; * MC-cluster-corrected <i>p</i><0.01.</p

Demographic characteristics for participants.

<p><i>Note</i>: n.s., not statistically significant at <i>p</i><0.05.</p