Citrate-Coated Magnetic Polyethyleneimine Composites for Plasmid DNA Delivery into Glioblastoma.

Several ternary composites that are based on branched polyethyleneimine (bPEI 25 kDa, polydispersity 2.5, 0.1 or 0.2 ng), citrate-coated ultrasmall superparamagnetic iron oxide nanoparticles (citrate-NPs, 8-10 nm, 0.1, 1.0, or 2.5 µg), and reporter circular plasmid DNA pEGFP-C1 or pRL-CMV (pDNA 0.5 µg) were studied for optimization of the best composite for transfection into glioblastoma U87MG or U138MG cells. The efficiency in terms of citrate-NP and plasmid DNA gene delivery with the ternary composites could be altered by tuning the bPEI/citrate-NP ratios in the polymer composites, which were characterized by Prussian blue staining, in vitro magnetic resonance imaging as well as green fluorescence protein and luciferase expression. Among the composites prepared, 0.2 ng bPEI/0.5 µg pDNA/1.0 µg citrate-NP ternary composite possessed the best cellular uptake efficiency. Composite comprising 0.1 ng bPEI/0.5 µg pDNA/0.1 µg citrate-NP gave the optimal efficiency for the cellular uptake of the two plasmid DNAs to the nucleus. The best working bPEI concentration range should not exceed 0.2 ng/well to achieve a relatively low cytotoxicity.

Evaluation of biocompatible alginate- and deferoxamine-coated ternary composites for magnetic resonance imaging and gene delivery into glioblastoma cells.

BACKGROUND: This paper describes comparative studies in cytotoxicities, magnetic resonance imaging (MRI), and gene delivery into glioblastoma U87MG or U138MG cells with ternary composites that are consist of superparamagnetic iron oxide (SPIO) nanoparticles (NPs) (size: 8-10 nm) with different surface coatings, circular plasmid DNA (pDNA) (~4 kb) equipped with fluorescent/luminescent probe, and branched polyethylenimine (25 kDa, PDI 2.5). METHODS: Three types of SPIO-NPs were used, including: (I) naked iron oxide NPs with Fe-OH surface group (Bare-NP); (II) iron oxide NPs with a coating of alginate (Alg-NPs); and (III) iron oxide NPs with a coating of deferoxamine (Def-NPs). By tuning the polyethylenimine (PEI)/NP ratios and with a fixed DNA amount, different ternary composites were employed for NP/gene transfection into glioblastoma U87MG or U138MG cells, which were then characterized by Prussian blue staining, in vitro MRI, green fluorescence protein (GFP) fluorescence and luciferase assay. RESULTS: Among the composites prepared, 0.2 ng PEI/0.5 µg DNA/1.0 µg Bare-NP ternary composite possessed the best cellular uptake efficiency of NP to the cytoplasm, following the trend Bare-NP > Alg-NP > Def-NP. This observation was consistent to the MRI assessments with in vitro T 2 relaxivity (r 2) values of 46.0, 35.5, and 23.7 s(-1)·µM(-1)·Fe, respectively. For cellular uptake efficiency of the pDNA, all variations of PEI/NP ratios of the composites did not yield significant differences. However, cellular uptake efficiencies of pDNA in the ternary composites in U138MG cells were generally higher than that of U87MG cells by an order of magnitude. Exceptionally, the ternary composite 0.2 ng PEI/0.5 µg DNA/1.0 µg Bare-NP possessed a lowered luciferase activity RLU for gene expression in U138MG cells. A total of 0.2 ng PEI/0.5 µg DNA/0.1 µg Bare-NP would be uptaken to the cell nucleus with the highest luciferase activity. A working concentration range of PEI with at least 15% higher cell viabilities than lipofectamine was 0.1 to 0.2 ng/well. The cytotoxicities became significant when 0.5 ng/well PEI was present in the ternary composites. CONCLUSIONS: The as-prepared composites offer potential biomedical applications in simultaneous gene delivery, imaging contrast enhancement, and metabolism study.

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells.

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca (2+)/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.

Nanoparticle-DNA-polymer composites for hepatocellular carcinoma cell labeling, sensing, and magnetic resonance imaging.

This paper describes comparative studies and protocols in (1) self-assembling of ultrasmall superparamagnetic iron oxide nanoparticle (NP), circular plasmid DNA, and branched polyethylenimine (PEI) composites; (2) magnetofection; (3) gene delivery, (4) magnetic resonance imaging (MRI), and (5) cytotoxicity of the composites toward hepatocellular carcinoma HepG2 cells.

Enhanced cellular uptake and gene delivery of glioblastoma with deferoxamine-coated nanoparticle/plasmid DNA/branched polyethylenimine composites.

Ternary composite nanomaterials based on deferoxamine-coated superparamagnetic iron oxide nanoparticles (8-10 nm), circular plasmid DNA (~4 kb) with fluorescent/luminescent reporter group, and branched polyethylenimine (25 kDa, PDI = 2.5) were prepared and compared in terms of their efficiencies in transfecting brain tumor cells at low concentration.

Ternary hybrid nanocomposites for gene delivery and magnetic resonance imaging of hepatocellular carcinoma cells.

This paper describes comparative studies in magnetic resonance imaging (MRI) and gene deliveries toward hepatocellular carcinoma (HCC) HepG2 cells with ternary composites that consist of superparamagnetic iron oxide (SPIO) nanoparticles (NPs) (8-10 nm) with deferoxamine coating, circular plasmid DNA (~4 kb) equipped with green fluorescent probe, and branched polyethylenimine (PEI) (25 kDa, PDI 2.5). The packaging of the ternary complexes has been characterized by agarose gel retardation assay. By tuning the PEI/NP ratios and with a fixed DNA amount, different ternary composites have been employed for NP/gene transfection towards HepG2 cells, which have been characterized by in vitro MRI and green fluorescence protein (GFP) fluorescence.

Photocytotoxicity and magnetic relaxivity responses of dual-porous γ-Fe2O3@meso-SiO2 microspheres.

Novel high magnetization microspheres with porous γ-Fe(2)O(3) core and porous SiO(2) shell were synthesized using a templating method, whereas the size of the magnetic core and the thickness of the porous shell can be controlled by tuning the experimental parameters. By way of an example, as-prepared γ-Fe(2)O(3)@meso-SiO(2) microspheres (170 nm) display excellent water-dispersity and show photonic characteristics under externally applied a magnetic field. The magnetic property of the γ-Fe(2)O(3) porous core enables the microspheres to be used as a contrast agent in magnetic resonance imaging with a high r(2) (76.5 s(-1) mM(-1) Fe) relaxivity. The biocompatible composites possess a large BET surface area (222.3 m(2)/g), demonstrating that they can be used as a bifunctional agent for both MRI and drug carrier. Because of the high substrate loading of the magnetic, dual-porous materials, only a low dosage of the substrate will be acquired for potential practical applications. Hydrophobic zinc(II) phthalocyanine (ZnPC) photosensitizing molecules have been encapsulated into the dual-porous microspheres to form γ-Fe(2)O(3)@meso-SiO(2)-ZnPC microspheres. Biosafety, cellular uptake in HT29 cells, and in vitro MRI of these nanoparticles have been demonstrated. Photocytotoxicity (λ > 610 nm) of the HT29 cells uptaken with γ-Fe(2)O(3)@meso-SiO(2)-ZnPC microspheres has been demonstrated for 20 min illumination.

Enhanced cellular uptake of aminosilane-coated superparamagnetic iron oxide nanoparticles in mammalian cell lines.

PURPOSE: To compare the cellular uptake efficiency and cytotoxicity of aminosilane (SiO(2)-NH(2))-coated superparamagnetic iron oxide (SPIO@SiO(2)-NH(2)) nanoparticles with three other types of SPIO nanoparticles coated with SiO(2) (SPIO@SiO(2)), dextran (SPIO@dextran), or bare SPIO in mammalian cell lines. MATERIALS AND METHODS: Four types of monodispersed SPIO nanoparticles with a SPIO core size of 7 nm and an overall size in a range of 7-15 nm were synthesized. The mammalian cell lines of MCF-7, MDA-MB-231, HT-29, RAW264.7, L929, HepG2, PC-3, U-87 MG, and mouse mesenchymal stem cells (MSCs) were incubated with four types of SPIO nanoparticles for 24 hours in the serum-free culture medium Dulbecco's modified Eagle's medium (DMEM) with 4.5 µg/mL iron concentration. The cellular uptake efficiencies of SPIO nanoparticles were compared by Prussian blue staining and intracellular iron quantification. In vitro magnetic resonance imaging of MSC pellets after SPIO labeling was performed at 3 T. The effect of each SPIO nanoparticle on the cell viability of RAW 264.7 (mouse monocyte/macrophage) cells was also evaluated. RESULTS: Transmission electron microscopy demonstrated surface coating with SiO(2)-NH(2), SiO(2), and dextran prevented SPIO nanoparticle aggregation in DMEM culture medium. MCF-7, MDA-MB-231, and HT-29 cells failed to show notable iron uptake. For all the remaining six cell lines, Prussian blue staining and intracellular iron quantification demonstrated that SPIO@ SiO(2)-NH(2) nanoparticles had the highest cellular uptake efficiency. SPIO@SiO(2)-NH(2), bare SPIO, and SPIO@dextran nanoparticles did not affect RAW 264.7 cell viability up to 200 µg Fe/mL, while SPIO@SiO(2) reduced RAW 264.7 cell viability from 10 to 200 µg Fe/mL in a dose-dependent manner. CONCLUSION: Cellular uptake efficiency of SPIO nanoparticles depends on both the cell type and SPIO surface characteristics. Aminosilane surface coating enhanced the cellular uptake efficiency without inducing cytotoxicity in a number of cell lines.

Synthesis of biocompatible, mesoporous Fe(3)O(4) nano/microspheres with large surface area for magnetic resonance imaging and therapeutic applications.

This article reports the fabrication of mesoporous Fe(3)O(4) nano/microspheres with a high surface area value (163 m(2)/g, Brunauer-Emmett-Teller) and demonstrates their use for drug loading, release, and magnetic resonance imaging (MRI). These monodispersed, mesoporous Fe(3)O(4) nano/microspheres with controllable average sizes ranging from 50 to 200 nm were synthesized using a Fe(3)O(4)/poly(acrylic acid) hybrid sphere template and subsequent silica shell formation and removal. We found that the SiO(2) coating is a crucial step for the successful synthesis of uniform mesoporous Fe(3)O(4) nano/microspheres. The as-synthesized mesoporous Fe(3)O(4) nanospheres show a high magnetic saturation value (M(s) = 48.6 emu/g) and could be used as MRI contrast agents (r(2) = 36.3 s(-1) mM(-1)). Trypan blue exclusion and MTT assay (see Supporting Information ) cytotoxicity analyses of the nanospheres based on HepG2 and MDCK cells showed that the products were biocompatible, with a lower toxicity than lipofectamine (positive control). Hydrophilic ibuprofen and hydrophobic zinc(II) phthalocyanine drug loading into mesoporous Fe(3)O(4) nanospheres and selected release experiments were successfully achieved. The potential use of mesoporous Fe(3)O(4) nanospheres in biomedical applications, in light of the nano/microspheres' efficient drug loading and release, MRI, and low cytotoxicity, has been demonstrated. It is envisaged that mesoporous Fe(3)O(4) nanospheres can be used as drug carriers and MRI contrast agents for the reticuloendothelial system; they can also be delivered locally, such as via a selective catheter.