3-3--dioctadecyloxacarbocyanine and nile-red

A novel and versatile design of DNA-lipid conjugates is presented. The assembly of the DNA headgroups into G-quadruplex structures is essential for the formation of micelles and their stability. By hybridization with a complementary oligonucleotide the micelles were destabilized, resulting in cargo release. In combination with a hairpin DNA aptamer as complementary strand, the release is obtained selectively by the presence of ATP.

Topics: Adenosine Triphosphate; Animals; Aptamers, Nucleotide; Carbocyanines; Cattle; DNA; Drug Carriers; Drug Liberation; Fluorescent Dyes; G-Quadruplexes; Lipids; Micelles; Nucleic Acid Hybridization; Oxazines; Serum Albumin, Bovine

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.

Topics: Acetone; Boron Compounds; Carbocyanines; Cells, Cultured; Chemical Precipitation; Flow Cytometry; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Lipids; Microalgae; Oxazines; Solvents; Staining and Labeling

A new micelle drug carrier that consists of a diblock polymer of propylene sulfide (PS) and N,N-dimethylacrylamide (poly(PS₇₄-b-DMA₃₁₀)) has been synthesized and characterized for site-specific release of hydrophobic drugs to sites of inflammation. Propylene sulfide was first polymerized using a thioacyl group transfer (TAGT) method with the RAFT chain transfer agent (CTA) 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (CEP), and the resultant poly(PS₇₄-CEP) macro-CTA was used to polymerize a second polymer block of DMA using reversible addition-fragmentation chain transfer (RAFT). The formation of the poly(PS₇₄-b-DMA₃₁₀) diblock polymer was confirmed by ¹H NMR spectra and gel permeation chromatography (GPC). Poly(PS₇₄-b-DMA₃₁₀) formed 100 nm micelles in aqueous media as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Micelles loaded with the model drugs Nile red and DiO were used to demonstrate the ROS-dependent drug release mechanism of these micelles following treatment with hydrogen peroxide (H₂O₂), 3-morpholinosydnonimine (SIN-1), and peroxynitrite. These oxidants were found to oxidize the micelle PPS core, making it more hydrophilic and triggering micelle disassembly and cargo release. Delivery of poly(PS₇₄-b-DMA₃₁₀) micelles dual-loaded with the Förster Resonance Energy Transfer (FRET) fluorophore pair DiI and DiO was used to prove that endogenous oxidants generated by lipopolysaccharide (LPS)-treated RAW 264.7 macrophages significantly increased release of nanocarrier contents relative to macrophages that were not activated. In vitro studies also demonstrated that the poly(PS₇₄-b-DMA₃₁₀) micelles were cytocompatible across a broad range of concentrations. These combined data suggest that the poly(PS₇₄-b-DMA₃₁₀) micelles synthesized using a combination of TAGT and RAFT have significant potential for site-specific drug delivery to tissues with high levels of oxidative stress.

Topics: Acrylic Resins; Animals; Carbocyanines; Cell Line; Cell Survival; Drug Carriers; Fluorescent Dyes; L-Lactate Dehydrogenase; Macrophages; Mice; Micelles; Oxazines; Reactive Oxygen Species; Sulfides