WO2013123934A1 - Procédé de fabrication ou de fusion de doubles couches de lipide planes - Google Patents

Procédé de fabrication ou de fusion de doubles couches de lipide planes Download PDF

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Publication number
WO2013123934A1
WO2013123934A1 PCT/DE2013/100051 DE2013100051W WO2013123934A1 WO 2013123934 A1 WO2013123934 A1 WO 2013123934A1 DE 2013100051 W DE2013100051 W DE 2013100051W WO 2013123934 A1 WO2013123934 A1 WO 2013123934A1
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WO
WIPO (PCT)
Prior art keywords
vesicles
solution
centrifuge
density
solid support
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Application number
PCT/DE2013/100051
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German (de)
English (en)
Inventor
Mira LISCHPER
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Nanospot Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanospot Gmbh filed Critical Nanospot Gmbh
Publication of WO2013123934A1 publication Critical patent/WO2013123934A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the invention relates to a method for the production of planar
  • Lipid bilayers on a solid support Lipid bilayers on a solid support.
  • a biological membrane serves to separate cells from an external medium as well as to form occluded regions and cell compartments within the cells.
  • a biological membrane essentially consists of a lipid bilayer and proteins arranged therein. The proteins fulfill different tasks. The exchange of substances through the membrane is made possible and regulated, for example, by transport proteins and channel proteins. In contrast, receptor proteins mediate stimuli or
  • Lipid bilayers are not only a component of natural, biological cell membranes, but are also artificially produced for scientific and pharmaceutical purposes.
  • lipid bilayers in the form of spherical, closed vesicles are used, the so-called liposomes.
  • In their interior can be pharmaceutical or cosmetic active substances
  • lipids can be included.
  • the nature and composition of the lipids can be tuned to precisely determine the site of drug release (so-called drug targeting). Different membranes can also fuse together. This plays e.g. a role in some viruses that infect cells via the fusion of their viral membrane with the membrane of the host cell, the process being mediated by viral fusion proteins.
  • the cell membrane is therefore the target for many pharmaceutical agents.
  • the exact understanding of the structure and function of membranes and membrane proteins is essential for the development of efficient therapeutic and diagnostic agents. Therefore, various model systems for biological membranes have been developed and investigated in recent years. These are for example
  • Lipid bilayers in the form of spherical vesicles with a diameter of ten nanometers so-called “small unilamellar vesicles” (abbreviated as SUVs), up to thirty microns in size “giant unilamellar vesicles” (abbreviated as GUVs) or so-called “large unilamellar vesicles” (US Pat.
  • SUVs small unilamellar vesicles
  • GUVs giant unilamellar vesicles
  • US Pat so-called “large unilamellar vesicles”
  • the vesicles or liposomes can either be freely bound in solution or bound to a solid support, in addition to the approximately spherical vesicles also flat, planar
  • Lipid bilayers so-called “supported lipid bilayers” (abbreviated as SLBs), which rest on a support, such as a glass slide or other substrate, can also be firmly attached to the support (so-called tethered bilayer lipid membranes) ", abbreviated as t-BLMs).
  • SLBs supported lipid bilayers
  • t-BLMs tethered bilayer lipid membranes
  • Lipid bilayers are suitable, these have established themselves as the most important model for natural cell membranes.
  • planar Usually arranged on a support, planar
  • Lipid bilayers ie SLBs (supported lipid bilayers), formed by adsorption of vesicles in solution on suitable surfaces, typically of SiO 2 , TiO 2 or mica.
  • suitable surfaces typically of SiO 2 , TiO 2 or mica.
  • Lipid compositions for spontaneous formation of SLBs for spontaneous formation of SLBs.
  • too high a cholesterol level in the vesicles can lead to the formation of SLBs Prevent SiO2 surfaces.
  • the formation of SLBs from vesicles of complex composition can be forced, for example, by the addition of Ca 2+ , osmotic stress, elevated temperature and long incubation times, a poor result is usually obtained because most vesicles on the surface have their spherical shape maintain and do not open.
  • Macromolecules in question including oligo (ethylene oxide), poly (ethylene oxide), poly (oxazoline) and oligopeptides with silanes or thiols as adhesion promoters.
  • membrane proteins are very sensitive and can easily denature under laboratory conditions, they must be stored in a lipophilic environment. This can be achieved by isolating the proteins from biological membranes and reconstituting them into artificial vesicles to form proteoliposomes. Then turn the proteins into planar ones
  • Proteoliposomes completely or partially absorbed by the planar lipid bilayer, so that the planar lipid bilayer then lipids and proteins of the liposomes.
  • One problem is that the merger must overcome energy barriers of both the vesicles and the receiving membrane.
  • One solution is that the vesicles and the receiving planar membrane have lipids with opposite charges.
  • Another possibility is the use of fusogenic peptides (SNAP-SNARE protein).
  • the pore-forming antibiotics nystatin and ergosterol may be added to the vesicles under specific osmotic conditions, permitting the vesicle membrane to permeate and the
  • the method comprises the following steps: a) one or more vesicles are prepared or provided b) the solid support is placed in a suitable centrifuge container in a centrifuge
  • Centrifuge container given wherein the vesicles have a higher density than the density of the solution, d) the centrifuge container with the vesicles and the
  • Solution is centrifuged until the vesicles on the support form a lipid bilayer.
  • a vesicle is to be understood as meaning approximately spherical bodies having an outer, unilamellar membrane in the form of a lipid bilayer.
  • the term "planar” is borrowed from the English term “planar bilayer” and has the meaning of planar or plan.
  • a planar lipid bilayer thus has a substantially uniformly flat surface, which is not curved in space.
  • the solid support is also essentially planar, but may also have structures, for example openings, elevations or measuring chambers.
  • a lipid bilayer is understood to be a membrane-like arrangement of lipids, as typically formed by phospholipids having a hydrophilic head and hydrophobic hydrocarbon tails in aqueous solution.
  • Lipid bilayer can be artificial lipids, natural lipids, synthetic
  • Lipid mixtures or natural lipid extracts each with or without reconstituted or naturally present protein.
  • the formation of a planar lipid bilayer from a vesicle occurs spontaneously on a solid support.
  • the vesicles must first attach to the solid support by diffusion and adsorption. Thereafter, the vesicle membrane must be broken, with the strong polar and entropic forces affecting the lipids in the membrane
  • the centrifugation step according to the invention now exerts a centrifugal force on the vesicles in the direction of the solid support.
  • the prerequisite for this is that the vesicles as a whole have a higher density than the density of the solution in which the vesicles are centrifuged.
  • the carrier is located at the bottom of the centrifuge container and is covered by the solution with the vesicles, the vesicles move by centrifugal force towards the carrier and adsorb to it.
  • the centrifuge container Since the solid support is planar and placed on the bottom of the centrifuge container, the centrifuge container must not be curved on the bottom so that the carrier is not damaged during centrifugation.
  • lipid bilayers can be prepared quickly and inexpensively on a solid support. For measurements, however, it is usually necessary in practice to reconstitute membrane proteins into an existing, planar lipid bilayer or others
  • the invention further relates to a method for the fusion of vesicles with planar lipid bilayers on a solid support, comprising the following steps: a) one or more vesicles are prepared or provided, b) the solid support having at least one planar
  • Lipid bilayer is in a suitable centrifuge container in a
  • the centrifugation step according to the invention exerts a centrifugal force on the vesicles. As a result, they move in the direction of the planar lipid bilayer, which is arranged on a support or rests on this. As a result of the further centrifugal force, the vesicles are opened when they strike the lipid bilayer and fuse with it.
  • the vesicles can be fused to a lipid bilayer prepared according to the prior art. Preferably, however, they are fused to a lipid bilayer prepared by the method of the invention on a solid support.
  • the vesicles must have a higher density than the solution in which the
  • Vesicles are centrifuged. This can be achieved by having the vesicles in their interior a solution whose density is higher than the density of the solution placed in the centrifuge container. This significantly increases the density of the vesicles overall.
  • the solution may be introduced into the vesicles by making them in the solution of increased density or by diluting the solution prior to centrifugation. Due to the increased density of the vesicles their sedimentation rate is greater and upon impact with the carrier or the lipid bilayer increase the forces acting. As a result, the vesicles or the lipid bilayers open more easily and the formation of planar lipid bilayers or their fusion with the vesicles is facilitated.
  • glycerol has the advantage that it is membrane-permeable and slow after centrifugation again diffused out of the formed and / or fused planar lipid bilayer.
  • liposomes or protein-enriched proteoliposomes can be used as vesicles.
  • GUVs giant unilamellar vesicles
  • LUVs large unilamellar vesicles
  • SUVs small unilamellar vesicles
  • Viral particles with a membrane are suitable. Also, biological membrane vesicles formed or made from natural biological membranes can be used. This provides a model system through which membrane proteins can be probed in their native lipid environment. This is a significant advantage because membrane proteins are very sensitive and may alter their conformation and behave differently in foreign lipid environments.
  • the solution introduced into the centrifuge container has at least one fusogenic substance, in particular divalent ions such as calcium chloride, destabilizing lipid membranes
  • Substances such as e.g. the pore-forming agents nystatin-cholesterol, complexing agents such as EDTA or SNARE proteins that regulate the fusion of biological membranes.
  • the fusogenic substances promote the formation and / or fusion of the planar lipid bilayer.
  • the advantage of the centrifugation steps is the use of a centrifuge with a "swing-out rotor.” As the rotor rotates, the rotor containers swing upwards until they are in a horizontal position, ensuring that the vector of centrifugal force
  • the solid support may be a planar glass plate, preferably a coverslip for microscopy, or a micro- or nanostructured measuring chip, which may be of any type for a micro- or nanometer-sized measuring chip
  • Nanostructuring suitable materials such as glass, silicon, silicon nitride, plastic or metals and recesses and / or protuberances in order to use these, for example, as measuring chambers for fluorescence measurements.
  • Liposomes are described in non-limiting examples.
  • Vesicle Prep Pro ® For the preparation of the commercially available, automated device "Vesicle Prep Pro ®” was used. By means of the device may solventless GUVs, thus “Giant unilamellar vesicles", are prepared according to the Electro Welling method with a diameter of 1-30 ⁇ .
  • the device has a chamber with an upper and a lower glass plate, between which the vesicle formation takes place. In order for the glass flakes to be electrically conductive, they are coated with indium tin oxide (abbreviated as "ITO").
  • ITO indium tin oxide
  • lipid film thus prepared on the ITO-coated glass slides was dried in a desiccator for one hour in the dark.
  • the lower glass plate with the lipid film was then inserted into the chamber of the "Vesicle Prep Pro ®" device.
  • the chamber was then filled with 640 ⁇ a solution in which pmol of a 6 kDa dextran were dissolved 150, corresponding to a concentration of about 1 mg
  • the solution had a higher density than the buffer solution with calcium chloride, in which the
  • the device was turned on, being between the ITO-coated
  • Lipid composition the solution and the desired amounts and size of the vesicles.
  • the chamber was opened and the upper glass plate carefully removed.
  • the formed GUVs were then removed with a pipette tip and placed as a solution in a test tube.
  • a commercial microscope coverslip was used, which was previously cleaned with solvents such as ethanol or propanol and then rinsed with distilled water and dried.
  • the coverslip was placed on the bottom of a centrifuge container and placed in the
  • Swing-out rotor also called “swing-out rotor" of a "Heraeus Megafuge 1 .0" centrifuge.
  • These rotors are not only suitable for coverslips, but also for other carriers, e.g. SBS microtiter plates.
  • the cover glass After centrifugation, the cover glass is largely covered with a stable planar lipid bilayer and can be used directly.
  • GUVs were prepared essentially as described above, with the difference that a lipid composition from 50% phosphatidylethanolamine (“PE”), 30% phosphatidylglycerol (“PG”) and 20% phosphatidycholine (“PC”).
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • PC phosphatidycholine
  • Lipid composition containing 40% phosphatidylethanolamine (“PE”), 30% phosphatidylglycerol (“PG”), 20% phosphatidycholine (“PC”) and 10% cardiolipin.
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • PC phosphatidycholine
  • the GUVs from both lipid compositions were filled with a sucrose solution inside to increase the density of the vesicles. Centrifugation was carried out for 30 minutes at 200 g in a solution to which 0.5 mmol calcium chloride had been added.
  • a lipid composition containing 80% diphytanoylphosphatidylcholine (“DPhPC”) and 20% cholesterol was prepared.
  • the GUVs prepared from this lipid composition were internally filled with a solution of approximately 1 mg / ml dextran to increase the density of the vesicles 40 minutes at 75 g in a solution to which 0.5 mmol calcium chloride was added.
  • FIGS. 1 a to 1 f show individual process steps for producing a lipid bilayer.
  • vesicles 1 are first prepared, ie liposomes of phospholipids, each having a hydrophilic head 3 and lipophilic
  • Hydrocarbon tails 4 have. These form an approximately spherical double layer with an interior or interior 2.
  • GUVs are produced, ie very large liposomes.
  • the production takes place in a medium in which, inter alia, substances 5 such as glycerol, sucrose or dextran are dissolved, whereby the density of the solution increases.
  • substances 5 such as glycerol, sucrose or dextran are dissolved, whereby the density of the solution increases.
  • This solution with the substance 5 is also present in the interior 2 of the vesicles 1.
  • Vesicles 1 and GUVs are then harvested and placed in a lower density buffer solution (not shown), i. the buffer solution does not contain the substances 5 or in a lower concentration.
  • the buffer solution also contains one or more fusogenic substances, for example
  • the buffer solution containing the vesicles 1 is then given a centrifuge container (not shown), at the bottom of which a structured measuring chip 100 is arranged as a solid carrier.
  • the measuring chip 100 points
  • the aperture of the measuring chambers 101 may be in the range of micrometers to a few nanometers as needed.
  • the centrifuge container is hung in or attached to a swinging bucket rotor (not shown) and the centrifuge is turned on.
  • Substances 5 because the double layer of the vesicles 1 is not permeable to the substances 5 or at least for the duration of the centrifugation, the substance 5 is not significantly effetlsäset. By this loading with the substance 5, the density of the vesicles 1 is increased in relation to the surrounding buffer solution. Due to the centrifugation, a strong force 6 is exerted on the vesicles 1, which acts orthogonally to the surface of the measuring chip 100 and accelerates the vesicles 1 in its direction.
  • FIG. 1 c shows that the vesicles 1 strike the measuring chip 100 after a certain centrifuging time, are compressed, lose their spherical structure and finally open from above.
  • FIG. 1 d shows that the vesicles 1 have completely opened on further centrifugation and have formed a planar lipid bilayer 7 on the measuring chip 100.
  • the measuring chamber 101 shown is completely covered by the planar lipid bilayer 7 and sealed at the top.
  • FIGS. 1 e and 1 f show how in the planar shown in Figure 1 d
  • Lipid bilayer 7 Membrane proteins 8 are incorporated by means of fusion.
  • first vesicles 10 are produced, which are smaller than GUVs, so-called LUVs (see above).
  • Membrane proteins 8 were reconstituted into the LUVs during production, so that they are proteoliposomes.
  • the smaller vesicles 10 are also filled or loaded in their interior 2 with substances 5, for example sucrose, which increase the density of the vesicles 10 in relation to the surrounding buffer solution (not shown) increase, in which the vesicles 10 are centrifuged.
  • the buffer solution also contains one or more fusogenic substances, for example calcium chloride.
  • the vesicles 10 are then centrifuged in the direction 6 of the measuring chip 100 where they fuse with the planar lipid bilayer 7 arranged on the measuring chip 100. As a result of the fusion, the membrane proteins 8 are arranged from the vesicles 10 in the lipid bilayer 7.
  • the measuring chip 100 is then removed from the centrifuge and can for
  • the membrane proteins 8 are, for example, a channel protein that transports a substrate (not shown) via the lipid bilayer 7, then the substrate in the
  • Measuring chamber 101 and can there by measurements for example
  • Fluorescence imaging can be detected quantitatively and time-resolved.
  • FIGS. 2a and 2b illustrate an example of the method in which a planar lipid bilayer 7 with membrane proteins 8 in only one
  • Centrifugation step can be made.
  • the sequence corresponds to
  • 2 of the vesicles V substances 5 are located inside, for example dextran, which increase the density of the vesicles V in relation to the surrounding buffer solution.
  • vesicles 1 used are GUVs into which membrane proteins 8 have been reconstituted during or after production. It are therefore proteoliposomes. Instead of GUVs but smaller proteoliposomes can be used, for example, LUVs, as shown in Figure 1 e.
  • LUVs LUVs
  • the measuring chip 100 can then be removed and the properties of the
  • Lipid membrane and membrane proteins can be directly examined with the above measuring methods.
  • the method described is particularly suitable for high throughput screenings and measurements.

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Abstract

L'invention concerne un procédé de fabrication ou de fusion de doubles couches de lipide planes (7) sur un support fixe (100). L'invention vise à permettre une fabrication rentable et efficace. A cet effet, le procédé comporte les étapes suivantes : a) des vésicules (1, 10) sont fabriquées ou produites et ajoutées dans une solution, les vésicules (1, 10) présentant une densité supérieure à la densité de la solution ; b) le support fixe (100) pourvu d'une double couche de lipide éventuelle (7) est disposé dans un contenant adapté dans une centrifugeuse ; c) la solution contenant les vésicules (1, 10) est versée dans le contenant de la centrifugeuse, de manière à ce que le support (100) soit recouvert par la solution ; d) la solution comprenant les vésicules (1, 10) est centrifugée, jusqu'à former une double couche de lipide (7) sur le support (100) ou à fusionner avec la double couche de lipide (7) éventuelle.
PCT/DE2013/100051 2012-02-23 2013-02-12 Procédé de fabrication ou de fusion de doubles couches de lipide planes WO2013123934A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE201210101480 DE102012101480A1 (de) 2012-02-23 2012-02-23 Verfahren zur Herstellung oder Fusion von planaren Lipiddoppelschichten
DE102012101480.9 2012-02-23

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WO2013123934A1 true WO2013123934A1 (fr) 2013-08-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058245A1 (fr) * 1997-06-17 1998-12-23 Molecular Metrology, Inc. Spectrometre a rayons x, dispersif selon l'angle
WO2004092394A2 (fr) * 2003-04-08 2004-10-28 The Children's Hospital Of Philadelphia Systeme de ligand avec ancrage membranaire a base de couche lipide plane, de type fluide, ayant une valence de ligand definie, et procedes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058245A1 (fr) * 1997-06-17 1998-12-23 Molecular Metrology, Inc. Spectrometre a rayons x, dispersif selon l'angle
WO2004092394A2 (fr) * 2003-04-08 2004-10-28 The Children's Hospital Of Philadelphia Systeme de ligand avec ancrage membranaire a base de couche lipide plane, de type fluide, ayant une valence de ligand definie, et procedes d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUSTO ET AL: "Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - BIOMEMBRANES, ELSEVIER, AMSTERDAM, NL, vol. 1768, no. 7, 11 June 2007 (2007-06-11), pages 1855 - 1860, XP022113278, ISSN: 0005-2736, DOI: 10.1016/J.BBAMEM.2007.04.025 *
NOEL A CLARK ET AL: "SURFACE-INDUCED LAMELLAR ORIENTATION OF MULTILAYER MEMBRANE ARRAYS", BIOPHYS. J, vol. 31, 1 July 1980 (1980-07-01), pages 65 - 95, XP055069145 *
OREOPOULOS J ET AL: "Combinatorial microscopy for the study of protein-membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM", JOURNAL OF STRUCTURAL BIOLOGY, ACADEMIC PRESS, UNITED STATES, vol. 168, no. 1, 1 October 2009 (2009-10-01), pages 21 - 36, XP026542229, ISSN: 1047-8477, [retrieved on 20090305], DOI: 10.1016/J.JSB.2009.02.011 *

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