Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with EGFR Mutations at a High Cell Density.

BACKGROUND/AIM: Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density. MATERIALS AND METHODS: We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities. RESULTS: The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-xL and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death. CONCLUSION: Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density.

Cyclic RGD-linked polymeric micelles for targeted delivery of platinum anticancer drugs to glioblastoma through the blood-brain tumor barrier.

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting αvß3/αvß5 integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, "cyclic-Arg-Ala-Asp" (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred via an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma.

Dependence of ion beam current on position of mobile plate tuner in multi-frequencies microwaves electron cyclotron resonance ion source.

We are constructing a tandem-type electron cyclotron resonance ion source (ECRIS). The first stage of this can supply 2.45 GHz and 11-13 GHz microwaves to plasma chamber individually and simultaneously. We optimize the beam current I(FC) by the mobile plate tuner. The I(FC) is affected by the position of the mobile plate tuner in the chamber as like a circular cavity resonator. We aim to clarify the relation between the I(FC) and the ion saturation current in the ECRIS against the position of the mobile plate tuner. We obtained the result that the variation of the plasma density contributes largely to the variation of the I(FC) when we change the position of the mobile plate tuner.

Profiles of ion beams and plasma parameters on a multi-frequencies microwaves large bore electron cyclotron resonance ion source with permanent magnets.

In order to contribute to various applications of plasma and beams based on an electron cyclotron resonance, a new concept on magnetic field with all magnets on plasma production and confinement has been proposed with enhanced efficiency for broad and dense ion beam. The magnetic field configuration consists of a pair of comb-shaped magnet surrounding plasma chamber cylindrically. Resonance zones corresponding for 2.45 GHz and 11-13 GHz frequencies are positioned at spatially different positions. We launch simultaneously multiplex frequencies microwaves operated individually, try to control profiles of the plasma parameters and the extracted ion beams, and to measure them in detail.

Electron cyclotron resonance plasma production by using pulse mode microwaves and dependences of ion beam current and plasma parameters on the pulse condition.

We measure the ion beam current and the plasma parameters by using the pulse mode microwave operation in the first stage of a tandem type ECRIS. The time averaged extracted ion beam current in the pulse mode operation is larger than that of the cw mode operation with the same averaged microwave power. The electron density n(e) in the pulse mode is higher and the electron temperature T(e) is lower than those of the cw mode operation. These plasma parameters are considered to cause in the increase of the ion beam current and are suitable to produce molecular or cluster ions.