n-n--4-xylylenebis(pyridinium) and pyranine

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.

Topics: Arylsulfonates; Carbocyanines; Color; Cytoplasm; Drug Carriers; Endocytosis; Endosomes; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Light; Liposomes; Membrane Fusion; Naphthalenes; Phosphatidylcholines; Phosphatidylethanolamines; Photolysis; Pyridinium Compounds; Sulfonic Acids; Temperature; Ultraviolet Rays

Endovesiculation by large unilamellar vesicles (LUVs) induced by cationic amphiphiles is described in this work. A recent procedure to monitor phagocytosis of vesicles by macrophages by determining the amount of the simultaneously internalized water_soluble fluorescent dye HPTS with external quencher was adapted to LUVs (Daleke, D. L.; Hong, K.; Papahadjopoulos, D. Biochim. Biophys. Acta 1990, 1024, 352). Compared to dibucaine and safingol, the local anesthetic chlorpromazine (CPZ) was found to be the most efficient inducer of HPTS-internalization by LUVs. Control experiments using LUVs with entrapped HPTS indicated that the observed dye-internalization does not originate from transient lysis. A strong increase in activity above the critical micelle concentration of CPZ implies the importance of CPZ-micelles for endovesiculation. The significantly less efficient CPZ-induced HPTS-internalization by LUVs with 68 nm compared to 176 nm diameter further diminishes the likelihood of a micelle/bilayer fusion mechanism and supports the presence of 'zipper-type' endovesiculation by LUVs with diameters as small as 68 nm.

Topics: Arylsulfonates; Chlorpromazine; Dibucaine; Dose-Response Relationship, Drug; Endocytosis; Fluorescent Dyes; Liposomes; Phosphatidylcholines; Pyridinium Compounds; Spectrum Analysis; Sphingosine

A delta pH 6 proton (internal pH 7.5, external pH 1.5) was imposed across the bilayer of egg phosphatidylcholine (PC) vesicles. The presence of the gradient was verified by the use of agents that predictably collapsed it. These agents included a detergent (Triton X-100), a pore-forming Na-H+ exchanger (nigericin), and weak acids capable of shuttling protons across the membrane (acetic and acetylsalicylic acids). The magnitude of the proton gradient and the rate of pH gradient collapse were determined by measuring pyranine fluorescence by use of an isosbestic point technique and calculating intravesicular pH from a calibration curve. Acid flux into the vesicles in the presence of chloride was measured directly by a simpler one-wavelength method. p-Xylene-bis-pyridinium bromide (DPX), a pyranine fluorescence quencher, was used to verify that the fluorescence signal originated from within the vesicles and that the observed rates of acidification were not an artifact due to pyranine leakage. Acid flux was found to be dependent on the ionic composition of the buffer. The presence of chloride ion in the external compartment caused a dramatic increase in the rate of acidification. It was demonstrated that the kinetics of acid flux into the vesicle were clearly controlled by the chloride-dependent mechanism at a relatively low chloride concentration of 2.5 mM. The model presented here is a simple and sensitive method for investigating the factors that influence acidification rates across membranes when acidification is driven by extremely large proton gradients, in the presence of chloride; conditions similar to those found in the stomach.

Topics: Acids; Arylsulfonates; Cell Membrane; Detergents; Fluorescent Dyes; Gastric Mucosa; Hydrogen-Ion Concentration; Lipid Bilayers; Models, Biological; Octoxynol; Phosphatidylcholines; Polyethylene Glycols; Protons; Pyridinium Compounds; Stomach