Journal Article

A549-luc-C8 cells; Bioware; Cell Line, Tumor; Cell Nucleus; Cell Separation; Electrophoresis; Humans; Microfluidic Analytical Techniques

This study reports a new biochip capable of cell separation and nucleus collection utilizing dielectrophoresis (DEP) forces in a microfluidic system comprising of micropumps and microvalves, operating in an automatic format. DEP forces operated at a low voltage (15 Vp-p) and at a specific frequency (16 MHz) can be used to separate cells in a continuous flow, which can be subsequently collected. In order to transport the cell samples continuously, a serpentine-shape (S-shape) pneumatic micropump device was constructed onto the chip device to drive the samples flow through the microchannel, which was activated by the pressurized air injection. The mixed cell samples were first injected into an inlet reservoir and driven through the DEP electrodes to separate specific samples. Finally, separated cell samples were collected individually in two outlet reservoirs controlled by microvalves. With the same operation principle, the nucleus of the specific cells can be collected after the cell lysis procedure. The pumping rate of the micropump was measured to be 39.8 microl/min at a pressure of 25 psi and a driving frequency of 28 Hz. For the cell separation process, the initial flow rate was 3 microl/min provided by the micropump. A throughput of 240 cells/min can be obtained by using the developed device. The DEP electrode array, microchannels, micropumps and microvalves are integrated on a microfluidic chip using micro-electro-mechanical-systems (MEMS) technology to perform several crucial procedures including cell transportation, separation and collection. The dimensions of the integrated chip device were measured to be 6x7 cm. By integrating an S-shape pump and pneumatic microvalves, different cells are automatically transported in the microchannel, separated by the DEP forces, and finally sorted to specific chambers. Experimental data show that viable and non-viable cells (human lung cancer cell, A549-luc-C8) can be successfully separated and collected using the developed microfluidic platform. The separation accuracy, depending on the DEP operating mode used, of the viable and non-viable cells are measured to be 84 and 81%, respectively. In addition, after cell lysis, the nucleus can be also collected using a similar scheme. The developed automatic microfluidic platform is useful for extracting nuclear proteins from living cells. The extracted nuclear proteins are ready for nuclear binding assays or the study of nuclear proteins.

Journal Article

Abscess; Animals; Bacterial Proteins; Bioware; Culture Media; Disease Models, Animal; DNA Ligases; Female; Gene Expression Regulation, Bacterial; Genes, Essential; Humans; Isopropyl Thiogalactoside; Mice; Mutation; Staphylococcal Skin Infections; Staphylococcus aureus; Xen29

A conditional expression system has been developed using the isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter to validate essential genes of Staphylococcus aureus in vivo. The system has been applied to prove the essentiality of ligA and to evaluate the function of tarI, which was found to be essential in vitro but not in vivo.

Journal Article

Animals, B16-F10-luc2, B16F10-luc2; Base Sequence; Cell Line, Tumor; Cholesterol/metabolism; Ethylenediamines/*chemistry; Fibrosarcoma/metabolism/pathology; Gene Silencing; Humans; Injections, Intravenous; Liposomes/administration & dosage/chemical; synthesis/*chemistry/pharmacokinetics; Male; Mice; Mice, Inbred BALB C; Molecular Imaging; Phosphoric Acid Esters/*chemistry; Polyethylene Glycols/chemistry; RNA, Small Interfering/genetics/*metabolism; Spectrophotometry, Infrared

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

Journal Article

IVIS, B16-F10-luc-G5, B16F10-luc-G5, B16-F10-luc, B16F10-luc,

Tumor progression is associated with the release of signaling substances from the primary tumor into the bloodstream. Tumor-derived cytokines are known to promote the mobilization and the recruitment of cells from the bone marrow, including endothelial progenitor cells (EPC). Here, we examined whether such paracrine influence could also influence the capacity of EPC to interfere with circulating metastatic cells. We therefore consecutively injected EPC pre-stimulated by tumor conditioned medium (CM-EPC) and luciferase-expressing B16 melanoma cells to mice. A net decrease in metastases spreading (vs non-stimulated EPC) led us to carry out a 2D-DIGE proteomic study to identify possible mediators of EPC-driven protection. Among 33 proteins exhibiting significant changes in expression, SPARC presented the highest induction after EPC exposure to CM. We then showed that contrary to control EPC, SPARC-silenced EPC were not able to reduce the extent of metastases when injected with B16 melanoma cells. Using adhesion tests and the hanging drop assay, we further documented that cell-cell interactions between CM-EPC and melanoma cells were promoted in a SPARC-dependent manner. This interaction led to the engulfment of melanoma cells by CM-EPC, a process prevented by SPARC silencing and mimicked by recombinant SPARC. Finally, we showed that contrary to melanoma cells, the pro-metastatic human breast cancer cell line MDA-MB231-D3H2 reduced SPARC expression in human EPC and stimulated metastases spreading. Our findings unravel the influence of tumor cells on EPC phenotypes through a SPARC-driven accentuation of macrophagic capacity associated with limitations to metastatic spread.

Journal Article

IVIS, B16-F10-luc-G5, B16F10-luc-G5, B16-F10-luc, B16F10-luc,

The therapeutic potential of tocotrienol, a vitamin E extract with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration. In this work, we demonstrated that novel transferrin-bearing, tocopheryl-based multilamellar vesicles entrapping tocotrienol significantly improved tocotrienol uptake by cancer cells overexpressing transferrin receptors. This led to a dramatically improved therapeutic efficacy in vitro, ranging from 17-fold to 72-fold improvement depending on the cell lines, compared to the free drug. In vivo, the intravenous administration of this novel tocotrienol formulation led to complete tumor eradication for 40% of B16-F10 murine melanoma tumors and 20% of A431 human epidermoid carcinoma tumors. Animal survival was improved by more than 20days compared to controls, for the two tumor models tested. These therapeutic effects, together with the lack of toxicity, potentially make transferrin-bearing vesicles entrapping tocotrienol a highly promising therapeutic system as part as an anti-cancer therapeutic strategy.