This work was supported by the Howard Hughes Medical Institute (HHMI) Collaborative Innovation Award. L. A. M. was supported by the Signaling and Cellular Regulation National Institute of Health training grant (T32 GM008759) and the NIH Ruth L. Kirschstein Pre-Doctoral Fellow (CA165349-01). We would like to thank Prof. Michael Stowell (CU Boulder), Prof. Douglas Rees and Prof. Rob Phillips (Caltech) for their invaluable comments.

1. Liposome Preparation
<ol>
	<li>Retrieve a 20 mL glass vial with a Teflon-lined cap.</li>
	<li>Clean all glassware and syringes with chloroform prior to use to prevent contamination.</li>
	<li>Transfer 100 &mu;L of reagent grade chloroform to the glass vial using a 250 &mu;L air-tight glass syringe.</li>
	<li>Add 30 &mu;L of reagent grade methanol to the same glass vial using a 100 &mu;L air-tight glass syringe.</li>
	<li>To prepare a 2 mM 7:1.5:1.5 phosphatidylcholine (POPC): phosphatidylethanolamine (POPE): cholest...

<ol>
	<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. 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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. 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No conflicts of interest declared.

Using the Avestin Liposofast LF-50 Extruder, we demonstrated how small-sized, synthetic liposomes are prepared through a pressure-controlled system. It is important to note that multilamellar vesicles form spontaneously following liposome hydration, which may lead to production of smaller nanoparticles. These small multilamellar vesicles will inevitably flow through the larger polycarbonate membrane pore size, causing heterogeneity in solutions of unilamellar vesicles produced by a large filter pore. Therefore, it is rec...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma-Aldrich</td>
 <td>02432-25ML</td>
 <td>95% stabilizers</td>
 </tr>
 <tr>
 <td>High Grade Methanol</td>
 <td>Sigma-Aldrich</td>
 <td>179337-4L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Liposofast LF-50 Extruder</td>
 <td>Avestin, Inc.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phospholipids</td>
 <td>Avanti Polar Lipids</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polycarbonate Pores</td>
 <td>Avestin, Inc.</td>
 <td>&nbsp;</td>
 <td>25 mm diameter</td>
 </tr>
 <tr>
 <td>Drain discs PE</td>
 <td>Avestin, Inc.</td>
 <td>230600</td>
 <td>25 mm diameter</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

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

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

constant-pressure-controlled-extrusion-method-for-preparation-nano

Research

JoVE Journal

Bioengineering

23.8K visualizações.  University of Colorado Boulder. Este protocolo descreve um método de extrusão para a preparação de vesículas lipídicas de sub-micron tamanhos com um elevado grau de homogeneidade. Este método utiliza um sistema de pressão controlada, com taxas de azoto controlados de fluxo para a preparação de lipossomas. A preparação de lípidos 1,2, Extrusão de lipossomas, e caracterização do tamanho será aqui contidas.