Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives.

Dihydroartemisinin (DHA) and artesunate (ARS), two artemisinin derivatives, have efficacious anticancer activities against human hepatocarcinoma (HCC) cells. This study aims to study the anticancer action of the combination treatment of DHA/ARS and farnesylthiosalicylic acid (FTS), a Ras inhibitor, in HCC cells (Huh-7 and HepG2 cell lines). FTS pretreatment significantly enhanced DHA/ARS-induced phosphatidylserine (PS) externalization, Bak/Bax activation, mitochondrial membrane depolarization, cytochrome c release, and caspase-8 and -9 activations, characteristics of the extrinsic and intrinsic apoptosis. Pretreatment with Z-IETD-FMK (caspase-8 inhibitor) potently prevented the cytotoxicity of the combination treatment of DHA/ARS and FTS, and pretreatment with Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited the loss of ΔΨm induced by DHA/ARS treatment or the combination treatment of DHA/ARS and FTS in HCC cells. Furthermore, silencing Bak/Bax modestly but significantly inhibited the cytotoxicity of the combination treatment of DHA/ARS and FTS. Interestingly, pretreatment with an antioxidant N-Acetyle-Cysteine (NAC) significantly prevented the cytotoxicity of the combination treatment of DHA and FTS instead of the combination treatment of ARS and FTS, suggesting that reactive oxygen species (ROS) played a key role in the anticancer action of the combination treatment of DHA and FTS. Similar to FTS, DHA/ARS also significantly prevented Ras activation. Collectively, our data demonstrate that FTS potently sensitizes Huh-7 and HepG2 cells to artemisinin derivatives via accelerating the extrinsic and intrinsic apoptotic pathways.

Hydrothermal Reduction of Polyethylenimine and Polyethylene Glycol Dual-Functionalized Nanographene Oxide for High-Efficiency Gene Delivery.

In this study, a physiologically stable dual-polymer-functionalized reduced nanographene oxide (nrGO) conjugate (PEG-nrGO-PEI, RGPP) with high efficiency of gene delivery is successfully synthesized through mixing PEGylated nanographene oxide (PEG-nGO, GP) and polyethylenimine (PEI, 25 kDa) solution under 80 °C for 2 h. This hydrothermal reduction of GP during PEIylation promotes the nucleophilic reaction between the amino moieties of PEI and the epoxy groups (or carboxylic groups) in GP and then forms C-NH- groups (or NH-CO groups) to covalently connect PEI and GP, which makes the RGPP nanocomposite more stable in physiological environments and has superior gene transfection efficiency compared with the nonhydrothermally reduced PEG-nGO/PEI conjugate (GPP) obtained by mixing GP and PEI under 20 °C for 2 h. Moreover, 808 nm laser irradiation (2 W/cm2) for 25 min increases ∼1.5-fold of gene transfection efficiency for RGPP but does not increase the gene transfection efficiency of GPP. Finally, RGPP is also able to efficiently deliver functional plasmid GFP-Bax (pGFP-Bax), exhibiting ∼43% of transfection efficiency in HepG2 cells. Collectively, the RGPP developed here is a highly efficient nanocarrier for gene delivery, and this work encourages further explorations of developing functionalized reduced nano-GO for high-efficiency gene therapy.

A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2.

A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02-0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe.

One-step reduction and PEIylation of PEGylated nanographene oxide for highly efficient chemo-photothermal therapy.

Graphene oxide (GO) has great potential in biomedical applications due to its excellent photothermal effect and drug loading. Herein, dual-functionalized reduced nano-GO (nrGO-PEG/PEI) has been developed as a synergistic system of drug delivery and an NIR-light-absorbing agent. Covalently PEGylated nanographene oxide (nGO-PEG) was simultaneously reduced and PEIylated by bathing with polyethylenimine (PEI 1.8 kDa) solution in water at 80 °C for 2 h to obtain nrGO-PEG/PEI. nrGO-PEG/PEI exhibited low cytotoxicity and excellent dispersibility in physiological environments. Furthermore, nrGO-PEG/PEI had ∼20-fold increment in NIR absorption at 808 nm and ∼2.7-fold increment in doxorubicin (DOX) loading over unreduced nGO-PEG. Loaded DOX could be efficiently released from nrGO-PEG/PEI/DOX in an acidic environment (pH 5.0) and by NIR irradiation. In addition, nrGO-PEG/PEI could be rapidly encapsulated into cells and targeted to the nucleus region. In vitro and in vivo studies demonstrated the remarkable anticancer effects of nrGO-PEG/PEI/DOX with NIR laser irradiation. These results suggest that nrGO-PEG/PEI may be a novel drug delivery platform for controlling chemo-photothermal synergistic cancer therapy.

Potent proapoptotic actions of dihydroartemisinin in gemcitabine-resistant A549 cells.

Our recent studies have demonstrated the key roles of reactive oxygen species (ROS)-mediated caspase-8- and Bax-dependent apoptotic pathways in dihydroartemisinin (DHA)-induced apoptosis of A549 cells. This report is designed to investigate the proapoptotic mechanisms of DHA in gemcitabine (Gem)-resistant A549 (A549GR) cells. A549GR cells exhibited lower basal antioxidant capacity, higher level of basal ROS and intracellular Fe(2+) than Gem-sensitive A549 (A549) cells. In contrast to the sluggish ROS generation induced by Gem, DHA induced a rapid ROS generation within 30min. Moreover, Gem induced similar ROS generation in both cell lines, while DHA induced more ROS generation in A549GR cells than in A549 cells. More importantly, after treatment with DHA, A549GR cells showed more potent induction in Bax activation, loss of mitochondrial membrane potential (ΔΨm), caspase activation and apoptosis than A549 cells. Furthermore, NAC pretreatment potently prevented DHA-induced ROS generation and loss of ΔΨm as well as apoptosis, and silencing Bax by shRNA or inhibition of one of caspase-3, -8 and -9 also significantly prevented DHA-induced apoptosis in both cell lines, indicating the key roles of ROS and Bax as well as the caspases. Collectively, DHA presents more potent proapoptotic actions in A549GR cells preferentially over normal A549 cells via ROS-dependent apoptotic pathway, in which Bax and caspases are involved.

One-step reduction and PEGylation of graphene oxide for photothermally controlled drug delivery.

Here, we developed one-step green reduction and PEGylation of nanosized graphene oxide (NGO) to obtain NrGO/PEG as a photothermally controllable drug delivery system. NrGO/PEG was synthesized by bathing methoxypolyethylene glycol amine (mPEG-NH2) and NGO at 90 °C for 24 h. The NrGO/PEG kept water stability for at least two months, and had ~14-fold increment in near-infrared (NIR) absorbance and ~2-fold increment in resveratrol (RV) loading over the unreduced NGO/PEG via π-π and hydrophobic interactions. Exposure of 4T1 cells to NrGO/PEG for 2 h showed 53.6% uptake ratio, and localization of NrGO/PEG in lysosomes instead of mitochondria. NIR irradiation (808 nm laser at 0.6 W/cm(2)) for 3 min potently enhanced RV release from NrGO/PEG-RV and the cytotoxicity of NrGO/PEG-RV against 4T1 cells, including decrease of cell viability, loss of mitochondrial membrane potential (ΔΨm) and cell apoptosis. Finally, NIR irradiation dramatically enhanced the efficacy of NrGO/PEG-RV in suppressing tumor growth in animal tumor models, further proving the remarkable synergistic action between photothermal effect of NrGO/PEG and RV loaded on NrGO/PEG.