carbocyanines 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

Surface plasmon coupled emission (SPCE) technique has attracted increasing attention in biomolecular interaction analysis and cell imaging because of its high sensitivity, low detection volume and low fluorescence background. Typically, the working range of SPCE is limited at nanometers to an interface. For micrometer-scale samples, new SPCE properties are expected because of complex coupling modes. In this work, cells with different subregions labeled were studied using a SPCE spectroscopy system. Angular and p-polarized emission was observed for cell membrane, cytoplasm, and nucleus labeled with DiI, Nile Red, and propidium iodide, respectively. The SPCE signals were always partially p-polarized, and the maximum emission angle did not shift, regardless of variations in emission wavelength, fluorophore distribution and stained layer thickness. Additionally, increased polarization and a broader angle distribution were also observed with an increase in sample thickness. We also investigated the impact of metallic substrates on the SPCE properties of cells. Compared with Au and Ni substrates, Al substrates presented better polarization and angle distribution. Moreover, the real-time detection of the cell labeling process was achieved by monitoring SPCE intensity. These findings expand SPCE from a surface technique to a 3D method for investigating bulk targets beyond the nanoscale interfaces, providing a basis to apply this technique to study cell membrane fluidity and biomolecule interactions inside the cell and to distinguish between cell subregions.

Topics: Carbocyanines; Computer Simulation; Feasibility Studies; Gold; HeLa Cells; Humans; Imaging, Three-Dimensional; Microscopy, Confocal; Models, Biological; Nickel; Oxazines; Polymethyl Methacrylate; Propidium; Surface Plasmon Resonance

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

Lipid nanoparticles and liposomal carrier systems are of growing interest for intravenous drug delivery due to their biocompatibility and targetability. It is, however, difficult to investigate their release behavior for lipophilic drugs under physiological conditions. This study describes a novel flow cytometric method studying drug transfer from such carrier systems to particles simulating physiological receptor sites. For this purpose, liquid and solid trimyristin nanoparticles or soybean phospholipid liposomes were loaded with the lipophilic fluorescent substances Nile red, temoporfin, and DiI. The transfer of these model drugs to large emulsion droplets was examined by flow cytometry. Transfer of DiI to differently sized acceptor emulsions was also monitored by separating donor and acceptor particles using ultracentrifugation. Flow cytometry revealed a completion of transfer within a few minutes for Nile red and temoporfin at considerable amounts of transferred dye. In contrast, the highly lipophilic DiI transferred over a period of weeks only for a small percentage of the dye. Ultracentrifugation results confirmed this for DiI and indicated a dependence of transfer characteristics on the acceptor surface area. Nile red transfer into a bulk oil phase as alternative acceptor system was also very slow. Flow cytometry seems to be well suited to study the intrinsic transfer of fluorescent lipophilic substances, as no kinetic hindrances like dialysis bags nor separation steps are required. Additional detailed experiments will, however, be necessary to elucidate the prevalent transfer mechanisms completely.

Topics: Carbocyanines; Drug Carriers; Emulsions; Flow Cytometry; Liposomes; Mesoporphyrins; Nanoparticles; Oxazines; Particle Size

Here we present the efficiency and versatility of newly developed core-multishell nanoparticles (CMS NPs), to encapsulate and transport the antitumor drugs doxorubicin hydrochloride (Dox), methotrexate (Mtx) and sodium ibandronate (Ibn) as well as dye molecules, i.e., a tetrasulfonated indotricarbocyanine (ITCC) and nile red. Structurally, the CMS NPs are composed of hyperbranched poly(ethylene imine) core functionalized by alkyl diacids connected to monomethyl poly(ethylene glycol). In order to evaluate their transport in aqueous media in vitro, we have used and compared SEC, UV, ITC, and NMR techniques. We observed that the CMS NPs were able to spontaneously encapsulate and transport Dox, Mtx and nile red in both organic and aqueous media as determined by SEC and UV-VIS spectroscopy. For the VIS transparent Ibn Isothermal Titration Calorimetric (ITC) experiments show an exothermic interaction with the CMS NPs. The enthalpic stabilization (DeltaH) upon encapsulation was in the order of approximately 7 kcals/mol which indicates stable interaction between Ibn and nanoparticles. A T(1) inversion recovery NMR experiment was carried out for 31P and 1H nuclei of Ibn and an increment of spin-lattice relaxation time for respective nuclei was observed upon encapsulation. CMS NPs were also found to encapsulate ITCC dye with stoichiometry of 6-8 molecules/nanocarrier. For in vivo imaging studies the dye loaded CMS NPs were injected to F9 teratocarcinoma bearing mice and a strong contrast was observed in the tumor tissues compared to free dye after 6 h of administration.

Topics: Animals; Antineoplastic Agents; Calorimetry; Carbocyanines; Chemistry, Pharmaceutical; Chromatography, Gel; Coloring Agents; Diphosphonates; Doxorubicin; Drug Carriers; Drug Compounding; Ibandronic Acid; Injections, Intravenous; Magnetic Resonance Spectroscopy; Methotrexate; Mice; Nanoparticles; Oxazines; Polyethylene Glycols; Polyethyleneimine; Spectrophotometry, Ultraviolet; Technology, Pharmaceutical; Teratocarcinoma

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.

Topics: Arteriosclerosis; Carbocyanines; Cell Differentiation; Cell Line; Cholesterol Esters; Filipin; Flow Cytometry; Fluorescent Dyes; Foam Cells; Humans; Lipoproteins, LDL; Macrophages; Oxazines; Receptors, LDL