Preparation of unilamellar liposomes of intermediate size (0.1–0.2 μm) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes

Szoka, Francis C.; Olson, Fred C.; Heath, Timothy D.; Vail, William J.; Mayhew, E.; Papahadjopoulos, Demetrios

doi:10.1016/0005-2736(80)90558-1

Cited by 470 publications

(190 citation statements)

References 19 publications

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“…Large unilamellar vesicles (LUVs) were prepared as described previously (34,35). Lipids were mixed in chloroform and dried as thin films under a nitrogen gas stream.…”

Section: Preparation Of Liposomesmentioning

confidence: 99%

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Song¹,

Sun²,

Coleman³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

106

135

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A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of M r 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix-hinge-helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.membrane-proximal ectodomain region ͉ neutralizing antibody ͉ vaccine design ͉ AIDS ͉ fusion T he 120-nm-diameter HIV-1 is a retrovirus that has a small genome consisting of 9 genes. The enveloped virion surface expresses the trimeric glycoprotein of M r 160,000 (gp160) spike protein, whose gp120 and gp41 subunits are assembled into noncovalently associated heterodimers. Unlike gp120, each gp41 subunit has a transmembrane segment (TM) that inserts into the membrane of the virus. The membrane proximal ectodomain region (MPER) links this TM to the folded gp41 ectodomain. Entry of HIV-1 into human T cells is mediated by attachment of the gp120 subunit to receptor CD4 and then binding to the coreceptor (CCR5 or CXCR4) (1). These interactions result in structural rearrangement of the gp41 subunit, followed by fusion of viral and host cell membranes (2, 3). Thus, antibody-mediated protection against HIV-1 must target the gp120/gp41 envelope trimer, the only viral protein exposed on the virion surface.Although high-titer, strain-specific neutralizing antibodies are readily generated during the course of natural infection or against subunit vaccines, broadly neutralizing antibodies (BNAbs), in contrast, are rarely produced (reviewed in ref. 4). Sequence variability, extensive glycosylation, and immunodominance of those exposed, largely variable segments subvert immune res...

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“…Large unilamellar vesicles (LUVs) were prepared as described previously (34,35). Lipids were mixed in chloroform and dried as thin films under a nitrogen gas stream.…”

Section: Preparation Of Liposomesmentioning

confidence: 99%

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Song¹,

Sun²,

Coleman³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

106

135

View full text Add to dashboard Cite

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“…In some experiments [3H]inulin (Amersham) was encapsulated in the vesicles as a metabolically inert marker of the aqueous contents [4, 51. All liposome preparations were sized by extrusion through 0.4 pm polycarbonate filters (Uni-Pore, Bio-Rad) [13] and freed from non-encapsulated marker on a Sephadex G-100 column [4].…”

Section: Liposomesmentioning

confidence: 99%

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro

Dijkstra

Galen

Regts

et al. 1985

European Journal of Biochemistry

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We investigated the intracellular metabolic fate of choline-labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline-phospholipid and phosphatidylserine (molar ration 5 : 4 : 1).With both labeled compounds only small proportions of water-soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell-associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of watersoluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine.With [Me-'4C]choline-labeled lysophosphatidylcholine, label was found to become cell-associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents..It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.

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“…Pure DOTMA liposomes were prepared in 150 mM-NaCI, 10 mM-TES (Sigma; Calbiochem) and 10 mM-sodium acetate pH 7.5 (fusion buffer). DOTMA liposomes for infectivity studies, and those containing a self-quenching concentration of Rho-PE (4 tool %) for binding and fusion studies, were prepared in NaCI/TES pH 7.5 by hydrating dried lipid (Bangham et al, 1965) and extruding three times under argon through double polycarbonate membranes of 0-08 or 0-2 ktm pore diameter (Poretics) in a Lipex Biomembranes high pressure extrusion apparatus, to achieve a uniform size distribution of vesicles (Szoka et al, 1980). This procedure results in the production of predominantly unilamellar vesicles (Diizgiine § et al, 1983;Hope et al, 1985).…”

Section: Methodsmentioning

confidence: 99%

Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: the role of CD4, serum and liposome-cell interactions

Konopka

Stamatatos

Larsen³

et al. 1991

Journal of General Virology

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Preparation of unilamellar liposomes of intermediate size (0.1–0.2 μm) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes

Cited by 470 publications

References 19 publications

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro

Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: the role of CD4, serum and liposome-cell interactions

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