Name: constant pressure-controlled extrusion method for the preparation of nano-sized lipid vesicles
Uploaded: 2012-06-22T13:00:00
Description: Watch this Scientific Journal Video about Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles at JoVE.com

Эта работа была поддержана Медицинского института Говарда Хьюза (HHMI) Совместные Innovation Award. LAM был поддержан сигнализации и клеточной регуляции Национального института здоровья подготовки гранта (T32 GM008759) и NIH Рут Л. Kirschstein предварительной защиты докторской диссертации (CA165349-01). Мы хотели бы поблагодарить профессора Майкла Стоуэлл (CU Boulder), профессор Дуглас Риз и профессор Роберт Филлипс (Caltech) за их неоценимую комментариев.

1. Подготовка липосом <ol><li> Получить 20 мл стеклянный флакон с тефлоновым покрытием крышки. </li><li> Очистите все посуду и шприцы с хлороформом перед использованием, чтобы предотвратить загрязнение. </li><li> Передача 100 мкл реагента хлороформ класса в стеклянном флаконе помощью 250 мкл гермети?...

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	<li>Connor, J., Bucana, C., Fidler, I. J., Schroit, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+expression+of+phosphatidylserine+in+mammalian+plasma+membranes:+quantitative+assessment+of+outer-leaflet+lipid+by+prothrombinase+complex+formation.">Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 3184-3188 (1989).</li><li>Smith, S. A., Morrissey, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+incorporation+of+tissue+factor+into+liposomes.">Rapid and efficient incorporation of tissue factor into liposomes.</a> J. Thromb. Haemost. 2, 1155-1162 (2004).</li><li>Hui, E., Johnson, C. P., Yao, J., Dunning, F. M., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin-mediated+bending+of+the+target+membrane+is+a+critical+step+in+Ca(2+)-regulated+fusion.">Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.</a> Cell. 138, 709-721 (2009).</li><li>Loughrey, H. C., Choi, L. S., Cullis, P. R., Bally, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedures+for+the+coupling+of+proteins+to+liposomes.">Optimized procedures for the coupling of proteins to liposomes.</a> J. Immunol. Methods. 132, 25-35 (1990).</li><li>Mui, B., Chow, L., Hope, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+technique+to+generate+liposomes+of+defined+size.">Extrusion technique to generate liposomes of defined size.</a> Methods Enzymol. 367, 3-14 (2003).</li><li>Gruner, S. M., Cullis, P. R., Hope, M. J., Tilcock, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+polymorphism:+the+molecular+basis+of+nonbilayer+phases.">Lipid polymorphism: the molecular basis of nonbilayer phases.</a> Annu. Rev. Biophys. Biophys. Chem. 14, 211-238 (1985).</li><li>Guven, A., Ortiz, M., Constanti, M., O'Sullivan, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+method+for+the+size+separation+of+homogeneous+fluorescein-encapsulating+liposomes.">Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes.</a> J. Liposome. Res. 19, 148-154 (2009).</li><li>Zimmerberg, J., Kozlov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+proteins+produce+cellular+membrane+curvature.">How proteins produce cellular membrane curvature.</a> Nature Reviews Molecular Cell Biology. 7, 9-19 (2006).</li><li>Chonn, A., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+liposomal+drug-delivery+systems.">Recent advances in liposomal drug-delivery systems.</a> Curr. Opin. Biotechnol. 6, 698-708 (1995).</li><li>Mayer, L. D., Hope, M. J., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicles+of+variable+sizes+produced+by+a+rapid+extrusion+procedure.">Vesicles of variable sizes produced by a rapid extrusion procedure.</a> Biochim. Biophys. Acta. 858, 161-168 (1986).</li><li>Matsuoka, K., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+liposomes+to+study+COPII-+and+COPI-coated+vesicle+formation+and+membrane+protein+sorting.">The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting.</a> Methods. 20, 417-428 (2001).</li><li>Dragovic, R. A., Gardiner, C., Brooks, A. S., Tannetta, D. S., Ferguson, D., Hole, P., Carr, B., Redman, C., Harris, A. L., Dobson, P. J., Harrison, P., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+and+phenotyping+of+cellular+vesicles+using+Nanoparticle+Tracking+Analysis.">Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis.</a> Nanomedicine. , (2011).</li><li>Hunter, D. G., Frisken, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+extrusion+pressure+and+lipid+properties+on+the+size+and+polydispersity+of+lipid+vesicles.">Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles.</a> Biophys J. 74, 2996-3002 (1998).</li></ol>

Использование Авестин Liposofast LF-50 Экструдер, мы продемонстрировали, как малогабаритные, синтетических липосом готовы через давление контролируемой системы. Важно отметить, что многослойные пузырьки образуют спонтанно после гидратации липосомы, которые могут привести к производству м?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Хлороформ </td><td> Sigma-Aldrich </td><td> 02432-25МЛ </td><td> 95% стабилизаторы </td></tr><tr><td> Высококачественный метанола </td><td> Sigma-Aldrich </td><td> 179337-4L </td><td></td></tr><tr><td> Liposofast LF-50 Экструдер </td><td> Авестин, Inc </td><td></td><td></td></tr><tr><td> Фосфолипиды </td><td> Avanti Полярные липиды </td><td></td><td></td></tr><tr><td> Поликарбонат поры </td><td> Авестин, Inc </td><td></td><td> Диаметром 25 мм </td></tr><tr><td> Слейте диски PE </td><td> Авестин, Inc </td><td> 230600 </td><td> Диаметром 25 мм </td></tr></tbody></table>

constant pressure-controlled extrusion method for the preparation of nano-sized lipid vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

Watch this Scientific Journal Video about Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles at JoVE.com

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Этот протокол описывает методом экструзии для подготовки липидные пузырьки субмикронных размеров с высокой степенью однородности. Этот метод использует давление управляемая система с регулируемым расходом азота в липосомы подготовки. Липидного подготовки<sup&gt; 1,2</sup&gt;, Липосомы экструзии, и размер характеристика будет представлена ​​в настоящем документе.

Постоянное давление контролируемый методом экструзии для приготовления наноразмерных липидных везикул

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

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