Two-Dimensional 16-BAC/SDS Polyacrylamide Gel Electrophoresis of Mitochondrial Membrane Proteins.

The substitution of the reverse polarity benzyldimethyl-n-hexadecylammonium chloride (16-BAC) polyacrylamide gel electrophoresis (PAGE) for isoelectric focusing (IEF) in the first dimension of electrophoresis improves the solubility of extremely hydrophobic proteins and their recovery compared to conventional 2D IEF/SDS PAGE. The acidic environment of 16-BAC PAGE has also been shown to better preserve the labile methylation of basic proteins such as the histones. Several improvements of the 2D 16-BAC/SDS PAGE method are collectively described here with particular emphasis on the separation of mitochondrial membrane proteins of low molecular mass. Lowering the 16-BAC concentration 50-fold in the gel and buffers decreases the formation of mixed 16-BAC/SDS micelles, which otherwise interferes with the separation of very low molecular mass proteins in second dimension SDS PAGE, and consequently improved the resolution of mitochondrial membrane proteins in the 10-30 kDa range.

Use of Adaptive Focused Acoustics™ ultrasound in controlling liposome formation.

Many techniques for producing large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) have drawbacks, including exposure of sensitive biological materials to harsh organic solvents or high temperatures. Here we describe the use of controlled focused ultrasound, Adaptive Focused Acoustics™ (AFA), to make LUV or SUV at low temperature without organic solvents and at a consistent, chosen size. We studied the effects of peak incident power (PIP), cycles per burst (CPB), duty factor (DF), temperature, and lipid composition (natural or synthetic), on liposome size distribution. We found that an increase in PIP, DF, CPB, or temperature decreased liposome size. When processed under the same conditions as the natural lipid composition [Phospholipon 90 G], the synthetic lipid composition [HSPC, DSPE-PEG-2000, Chol] generally produced larger liposomes, although extending processing time reduced liposomes to similar size. In combination with AFA, these trends can help pinpoint parameter values that achieve a desired liposome size distribution.

Crude Garlic Extract Inhibits Cell Proliferation and Induces Cell Cycle Arrest and Apoptosis of Cancer Cells In Vitro.

Garlic and its lipid-based extracts have played an important medicinal role in humans for centuries that includes antimicrobial, hypoglycemic, and lipid-lowering properties. The present study was to investigate the effects of crude garlic extract (CGE) on the proliferation of human breast, prostate, hepatic, and colon cancer cell lines and mouse macrophageal cells, not previously studied. The human cancer cell lines, such as hepatic (Hep-G2), colon (Caco-2), prostate (PC-3), and breast (MCF-7), were propagated at 37°C; air/CO2 (95:5 v/v) using the ATCC-formulated RPMI-1640 Medium and 10% fetal bovine serum (FBS), while the mouse macrophage cell line (TIB-71) was propagated at 37°C; air/CO2 (95:5 v/v) using the ATCC-formulated DMEM and 10% FBS. All cells were plated at a density of ∼5000 cells/well. After overnight incubation, the cells were treated with 0.125, 0.25, 0.5, or 1 µg/mL of CGE an additional 72 h. Inhibition of cell proliferation of 80-90% was observed for Hep-G2, MCF-7, TIB-71, and PC-3 cells, but only 40-55% for the Caco-2 cells when treated with 0.25, 0.5, or 1 µg/mL. In a coculture study of Caco-2 and TIB-71 cells, inhibition of cell proliferation of 90% was observed for Caco-2 cells compared to the 40-55% when cultured separately. CGE also induced cell cycle arrest and had a fourfold increase in caspase activity (apoptosis) in PC-3 cells when treated at a dose of 0.5 or 1 µg/mL. This investigation of CGE clearly highlights the fact that the lipid bioactive compounds in CGE have the potential as promising anticancer agents.

A nanoemulsion formulation of dacarbazine reduces tumor size in a xenograft mouse epidermoid carcinoma model compared to dacarbazine suspension.

Dacarbazine (DAC) is an anticancer drug that has been used to treat various types of cancers. The aim of the current study was to test whether there is an increased efficacy of DAC as a nanoemulsion on reducing tumor size in an epidermoid carcinoma xenograft mouse model. Tumors were induced in 5-week-old nude mice by subcutaneous injection. The mice were untreated or treated with a suspension of DAC (0.1 mg/kg), a nanoemulsion of DAC (0.1 mg/kg), or Nano-Control (same composition as the suspension and nanoemulsion but no DAC), every 2 days by either intramuscular injection (IM) or topical application. After 40 days, the final tumor size of mice receiving the nanoemulsion of DAC IM (0.83 ± 0.55 mm(3)) was significantly reduced compared to the suspension of DAC IM (4.75 ± 0.49 mm(3)), Nano-Control IM (7.63 ± 0.91 mm(3)), and untreated (10.46 ± 0.06 mm(3)). The final tumor size of mice receiving the nanoemulsion of DAC topically (3.33 ± 0.63 mm3) was also significantly reduced compared to the suspension of DAC topically (7.64 ± 0.68 mm(3)). This increased efficacy maybe partially attributed to: 1) the reduced particle size of the nanoemulsion in comparison with suspension (111 versus > 6000 nm), 2) reduction in zeta potential of the nanoemulsion compared to suspension (-3.2 versus -89.1 mV), 3) production of a stable water dispersion relative to unstable suspension, 4) decreased polydispersity index of the nanoemulsion compared to suspension, and 5) greater stability of drug with the nanoemulsion in comparison with the suspension. FROM THE CLINICAL EDITOR: In this clinically relevant study, the anti-tumor efficacy of dacarbazine was found to be significantly increased as a nanoemulsion in epidermoid carcinoma xenograft mice, both with IM and topical administration.

A nanoemulsion formulation of tamoxifen increases its efficacy in a breast cancer cell line.

This paper reports on the preparation of a water-soluble nanoemulsion of the highly lipid-soluble drug tamoxifen (TAM). In addition, relative to a suspension of TAM, the nanoemulsion preparation demonstrated a greater zeta potential (increased negative charge) which has previously been associated with increasing drug/membrane permeability. This study also reports that relative to suspensions of TAM with particle sizes greater than 6000 nm, nanoemulsions of TAM, having mean particle sizes of 47 nm, inhibited cell proliferation 20-fold greater and increased cell apoptosis 4-fold greater in the HTB-20 breast cancer cell line. Thus, this work suggests that a nanoemulsion compared to a suspension preparation of TAM increases its anticancer properties relative to breast cancer.

Nanoemulsion preparations of the anticancer drug dacarbazine significantly increase its efficacy in a xenograft mouse melanoma model.

This article reports on the preparation of a water-soluble nanoemulsion of the highly lipid-soluble drug Dacarbazine (DAC). In addition, relative to suspensions of DAC, the nanoemulsion preparation demonstrated a lower zeta-potential (decreased negative charge, less anionic and more cationic) which has previously been associated with influencing drug membrane permeability. This study also reports that, relative to suspensions of DAC with a mean particle size of 5470 nm, nanoemulsions of DAC having mean particle sizes of 131 nm were more efficacious. For example, in a mouse xenograft model using a human melanoma cell line, a topical application of nanoemulsions of DAC compared to the suspension preparation of DAC produced up to 10-fold greater percent (%) reductions of tumor size. The reduction in tumor size by the intramuscular (IM) injection (-61%) and topical application of the nanoemulsion preparations of DAC (-49%) appeared to be comparable in efficacy, although the former was statistically greater (p < 0.05). In addition, 12 weeks after DAC treatment cessation, 98% of the animals given the IM application of the nanoemulsion of DAC remained tumor-free compared to the control or untreated animals. During this drug cessation period, and compared to the suspension preparations, nanoemulsions of DAC showed 5-fold greater efficacies (73% versus 14%) in preventing tumor growth. In conclusion, in this xenograft mouse model of melanoma, nanoemulsion suspensions of DAC are more efficacious in the treatment and prevention of tumor growth.