A simple method for the preparation of homogeneous phospholipid vesicles

Barenholz, Yechezkel; Gibbes, D.C.; Bj, Litman; Goll, Johannes; Te, Thompson; Rd, Carlson

doi:10.1021/bi00631a035

Cited by 877 publications

(474 citation statements)

References 19 publications

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“…This can be compensated by ultracentrifugation, if highly concentrated liposomes are needed. During ultracentrifugation the liposomes sediment and the supernatant can be removed easily 27 . Other ways to concentrate liposomes such as by dialysis 28 are more time consuming than ultracentrifugation.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for <em>In Vivo</em> Optical Imaging

Tansi

Rüger

Rabenhold

et al. 2015

JoVE

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Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed LipdQ) and the free DY-676-COOH were compared with Lip-Q in animal trials. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for <em>In Vivo</em> Optical Imaging

Tansi

Rüger

Rabenhold

et al. 2015

JoVE

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“…All NMR experiments were performed with pulsed-field gradient enhanced pulse sequences (15,16) Miscellaneous-SUVs of 100% brain phosphatidylcholine or 30% brain phosphatidylserine, 70% brain phosphatidylcholine (all from Avanti Polar Lipids) were made and quantified as described (17). SUVs were confirmed to be unilamellar by electron microscopy.…”

Section: Methodsmentioning

confidence: 99%

A Broken α-Helix in Folded α-Synuclein

Chandra

Chen

Rizo

et al. 2003

Journal of Biological Chemistry

517

587

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␣-Synuclein is a small cytosolic protein of presynaptic nerve terminals composed of seven 11-residue repeats and a hydrophilic tail. ␣-Synuclein misfolding and dysfunction may contribute to the pathogenesis of Parkinson's disease and neurodegenerative dementias, but its normal folding and function are unknown. In solution, ␣-synuclein is natively unstructured but assumes an ␣-helical conformation upon binding to phospholipid membranes. We now show that this conformation of ␣-synuclein consists of two ␣-helical regions that are interrupted by a short break. The structural organization of the ␣-helices of ␣-synuclein was not anticipated by sequence analyses and may be important for its pathogenic role.In recent years, the presynaptic protein ␣-synuclein has attracted much attention because of its involvement in neurodegenerative diseases (1-3). Two independent mutations in human ␣-synuclein cause familial Parkinson's disease, and wild type ␣-synuclein is a major component of Lewy bodies, cytoplasmic inclusion bodies found in Parkinson's disease and in several forms of neurodegenerative dementia. However, independent of its role in neurodegenerative diseases, ␣-synuclein is an interesting protein in its own right. It is an abundant presynaptic protein that may regulate neurotransmitter release and may contribute to synaptic plasticity (4 -6). ␣-Synuclein is the founding member of a protein family that additionally includes ␤-and ␥-synucleins and synoretin (7-9). The sequences of all synucleins are similar, although only ␣-synuclein is implicated in disease. Synucleins are composed of six copies (␤-synuclein) or seven copies (all other synucleins) of an unusual 11-residue imperfect repeat, followed by a variable short hydrophilic tail. Synucleins are soluble, natively unfolded proteins that avidly bind to negatively charged phospholipid membranes and become ␣-helical upon binding (10). Although secondary structure predictions indicate that the synuclein repeats could form an amphipathic structure consistent with lipid binding, the ␣-helical conformation is puzzling because the synuclein repeats are punctuated by central glycine residues. Furthermore, in Lewy bodies ␣-synuclein is thought to be in a ␤-strand aggregate, but aggregation of ␣-synuclein into dimers and multimers is promoted by lipid environments that induce an ␣-helical conformation (11-13). In the present study, we have examined the conformation of ␣-synuclein in lipidic environments to understand the relation of its sequence to its physicochemical properties and to map a potential pathway of misfolding in neurodegenerative disease. EXPERIMENTAL PROCEDURESProduction of ␣-Synuclein-Recombinant ␣-synuclein was expressed in bacteria as GST-fusion proteins with a TEV protease recognition sequence preceding the N-terminal methionine and cleaved with TEV protease (Invitrogen), resulting in a single additional glycine residue at the N terminus. After TEV cleavage, ␣-synuclein was isolated as the only heat-stable component upon boiling for 15 min, purified by i...

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“…DOTAP/CHOL and DOTAP/DOPE lipoplexes were prepared (Table 2) by mixing 0-21.56 nmol cationic lipid with 3.08 nmol DNA (0-7.0 L + /DNA Ϫ mole ratio). 47 charge ratio of 88:1 DNA Ϫ /protamine sulfate + ) for 10 min at room temperature and then adding DOTAP/CHOL liposomes (0-6.0 L + /DNA Ϫ mole ratio). 19,20 DOTAP/CHOL-spermidine complexes were prepared (Table 2) by incubating 3.08 nmol DNA and 0.5 nmol spermidine (containing three positive charges per molecule, two primary and one secondary amino groups, (6.16:1 DNA/spermidine mole ratio) for 10 min at room temperature and then adding DOTAP/CHOL liposomes (0-6.0 L + /DNA Ϫ mole ratio).…”

Section: Lipoplex Preparationmentioning

confidence: 99%

“…47 Conductivity measurements were Gene Therapy done in a V-TECH Oyster pH, temperature, conductivity meter (St Louis, MO, USA) at 37°C. Osmolality measurements were done in a WESCOR Vapor Pressure Osmometer 550 (Logan, UT, USA).…”

Section: Characterization Of Mediamentioning

confidence: 99%