DE102012101480A1 - Process for the preparation or fusion of planar lipid bilayers - Google Patents

Abstract

The invention relates to a method for the production or fusion of planar lipid bilayers (7) on a solid support (100). In order to enable cost-effective and efficient production, the following method is proposed: a) vesicles (1, 10) are prepared or prepared and placed in a solution, the vesicles (1, 10) having a density higher than the density of the solution, b) the solid support (100) with an optional lipid bilayer (7) is placed in a suitable centrifuge container in a centrifuge, c) the solution containing the vesicles (1, 10) is placed in the centrifuge container so that the support (100) d) the solution containing the vesicles (1, 10) is centrifuged until they form a lipid bilayer (7) on the carrier (100) or fuse with the optional lipid bilayer (7).

Description

The invention relates to a method for producing planar lipid bilayers on a solid support.

Biological membranes serve 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. On the other hand, receptor proteins mediate stimuli or signals, for example through extracellular ligand binding, which then leads to secondary processes intracellularly.

Lipid bilayers are not only a component of natural, biological cell membranes, but are also artificially produced for scientific and pharmaceutical purposes. For this purpose, lipid bilayers in the form of spherical, closed vesicles are used, the so-called liposomes. In its interior, pharmaceutical or cosmetic active ingredients may 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 micrometers in size "giant unilamellar vesicles" (abbreviated as GUVs) or so-called "large unilamellar vesicles "(abbreviated as LUVs) whose size lies approximately in between. The vesicles or liposomes can either be free in solution or bound to a solid support. In addition to the approximately spherical vesicles also flat, planar lipid bilayers are used, so-called "supported lipid bilayers" (abbreviated as SLBs), which rest on a support, such as a glass plate or other substrate. The planar lipid bilayers may also be firmly attached to the support (so-called "tethered bilayer lipid membranes", abbreviated as t-BLMs).

Since biological membranes vary considerably in composition, different model systems have been developed. The difficulty lies in making the artificial model systems as close as possible to the complex biological membranes and, on the other hand, simplifying them so that the structure and functionality of individual components can be investigated. Because many modern research methods such as scanning probe microscopy, impedance spectroscopy, fluorescence imaging, quartz crystal microbalance (QCM), electrophysiological measurements, and surface plasmon resonance spectroscopy are particularly suited for planar lipid bilayers, these have become established as the most important model for natural cell membranes.

Usually, planar lipid bilayers arranged on a support, ie SLBs (supported lipid bilayers), are formed by adsorption of vesicles in solution on suitable surfaces, typically of SiO ₂ , TiO ₂ or mica. However, only certain conditions and lipid compositions lead to spontaneous formation of SLBs. For example, too high a cholesterol level in the vesicles can prevent the formation of SLBs on SiO ₂ surfaces. Although 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.

Another approach is to make planar membranes tethered to a solid support (tethered bilayer lipid membranes). For this purpose, so-called spacers are used which are covalently bonded at one end to the hydrophilic lipid heads and at the other end to the solid support. Suitable spacers are numerous linear macromolecules, including oligo (ethylene oxide), poly (ethylene oxide), poly (oxazoline) and oligopeptides with silanes or thiols as adhesion promoters.

Since 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 making the proteins from biological Membranes are reconstituted and reconstituted into artificial vesicles to form proteoliposomes. To then introduce the proteins into planar lipid bilayers, the proteoliposomes are fused with a planar membrane. In a fusion, therefore, liposomes or proteoliposomes are completely or partially taken up by the planar lipid bilayer, so that the planar lipid bilayer then has lipids and proteins of the liposomes. One problem, however, 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). Alternatively, the pore-forming antibiotics nystatin and ergosterol may be added to the vesicles under specific osmotic conditions, permitting the vesicle membrane to permeabilize and increasing the likelihood of fusion.

All of these known methods are time-consuming and technically very complicated and inefficient, since a significant proportion of the proteins are not incorporated into the planar membranes and are lost. Furthermore, to date, no method is known to produce SLBs directly from biological cell membranes to study membrane proteins in their native lipid environment.

The object of the invention is therefore to propose a method that does not have the disadvantages mentioned and enables a cost-effective and efficient production of planar membranes or lipid bilayers on a solid support.

This object is achieved in that 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, c) the vesicles are placed in the centrifuge container together with a solution, the vesicles having a higher density than the density the solution, d) centrifuge the centrifuge container with the vesicles and the solution until the vesicles on the carrier form a lipid bilayer.

For the purposes of the invention, 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. For the preparation of vesicles and / or planar lipid bilayer, artificial lipids, natural lipids, synthetic lipid mixtures or natural lipid extracts, each with or without reconstituted or naturally present protein, may be used.

In the prior art, the formation of a planar lipid bilayer from a vesicle occurs spontaneously on a solid support. In this process, the vesicles must first attach to the solid support by diffusion and adsorption. Thereafter, the vesicle membrane must be disrupted, overcoming the strong polar and entropic forces that hold the lipids together in the membrane. This energetically unfavorable or unlikely process requires a lot of time and is technically complex.

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. Procedural steps to increase the density of the vesicles are described below. Since 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. As a result of the further centrifugal force, the vesicles are opened and a planar lipid bilayer forms on the support. 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.

By the method described above, 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 to introduce other substances or to alter the lipid composition. To achieve this object, 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 incorporated in a suitable centrifuge container arranged in a centrifuge, c) the vesicles are added together with a solution in the centrifuge container, d) the centrifuge container with the vesicles and the solution is centrifuged until the vesicles fuse with the lipid bilayer on the carrier.

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.

Advantageous embodiments of the invention with additional process features are described below.

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.

It has been found that glycerol, dextran, sucrose, other sugars, salts or nanoparticles are suitable for increasing the density. Glycerol, inter alia, has the advantage that it is membrane-permeable and, after centrifugation, slowly diffuses out again from the formed and / or fused planar lipid bilayer.

As vesicles, liposomes or protein-enriched proteoliposomes can be used. In particular, GUVs ("giant unilamellar vesicles"), LUVs ("large unilamellar vesicles"), SUVs ("small unilamellar vesicles") or virus 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.

A further improvement is possible if the solution added to the centrifuge container has at least one fusogenic substance, in particular bivalent ions such as calcium chloride, substances destabilizing lipid membranes 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.

Advantageous for the centrifugation steps is the use of a centrifuge with a swing-out rotor ("swing-out rotor"). As the rotor rotates, the rotor containers swing upward until they are in a horizontal position. This ensures that the vector of centrifugal force extends radially from the rotor axis and orthogonal to the plane of the solid support.

The solid support may be a planar glass slide, preferably a coverslip for microscopy. Also particularly suitable is or a micro- or nanostructured measuring chip. This may consist of all suitable for a micro- or nanostructuring materials such as glass, silicon, silicon nitride, plastic or metals and have depressions and / or protuberances to use them, for example, as measuring chambers for fluorescence measurements.

In particular, with the mentioned measuring chips, the lipid bilayers produced can be measured by means of fluorescence imaging, scanning probe microscopy, impedance spectroscopy, quartz crystal microbalances (QCM for short), surface plasmon resonance spectroscopy or electrophysiologically.

The preparation of planar lipid bilayers of liposomes is described below in non-limiting examples.

For the preparation of the commercially available, automated device "Vesicle Prep Pro ^®" was used. By means of the device solvent-free GUVs, so "Giant unilamellar vesicles", be prepared with a diameter of 1 to 30 microns by the electroswelling method. 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").

First, 10 μl of the desired lipids were dissolved in chloroform and evenly distributed with a pipette tip on the ITO-coated sides of the glass slides. As the lipid, diphytanoylphosphatidylcholine ("DPhPC") was used. The 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 placed the device into the chamber of the "Vesicle Prep Pro ^®". The chamber was then filled with 640 μl of a solution in which 150 pmol of a 6 kDa dextran was dissolved, which corresponds to a concentration of approximately 1 mg / ml. As a result, the solution had a higher density than the calcium chloride buffer solution in which the centrifugation was carried out. The second, upper ITO-coated glass slide was then inserted into the chamber and the chamber sealed with a teflon ring and grease.

The device was switched on, with a current flowing between the ITO-coated glass plates, which act as electrodes. As a result, the GUVs begin to form. The formation of the liposomes can take between a few hours and 18 hours. The time required depends on the type of lipid composition, the solution and the desired amounts and size of the vesicles.

After switching off the device, 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.

As a solid support, 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-bucket 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. Forty μl of the GUV-containing solution was then added to a buffer solution which i.a. Calcium chloride in a concentration of 0.5 mmol / l contained. The calcium chloride improves the later spreading of the vesicles on the surface and the formation of the planar lipid bilayer. Thereafter, the solution was centrifuged at 75 g for 40 minutes. With other composition of the lipids and the solutions used, the centrifugation parameters must be adjusted accordingly.

After centrifugation, the cover glass is largely covered with a stable planar lipid bilayer and can be used directly.

In one variant of the method, GUVs were prepared essentially as described above, except that a lipid composition of 50% phosphatidylethanolamine ("PE"), 30% phosphatidylglycerol ("PG") and 20% phosphatidycholine ("PC") was used ,

In another variant of the method, the GUVs were prepared from a lipid composition containing 40% phosphatidylethanolamine ("PE"), 30% phosphatidylglycerol ("PG"), 20% phosphatidycholine ("PC") and 10% cardiolipin.

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.

In another variant of the method, the GUVs were prepared from a lipid composition containing 80% diphytanoyl phosphatidylcholine ("DPhPC") and 20% cholesterol. The GUVs prepared from this lipid composition were filled internally with a solution of about 1 mg / ml dextran to increase the density of the vesicles. The centrifugation was carried out for 40 minutes at 75 g in a solution to which 0.5 mmol calcium chloride was added.

The invention will be described with reference to a drawing by way of example and not limitation, with further advantageous details of the figures of the drawing can be seen. Genus or functionally identical objects are provided with the same reference numerals. The drawings and description are illustrative and are not meant to be quantitative. All objects are shown schematically and not true to scale.

The 1a to 1f show individual process steps for producing a lipid bilayer. For this purpose, first vesicles 1 produced, ie liposomes of phospholipids, each having a hydrophilic head 3 and lipophilic hydrocarbon tails 4 exhibit. These form an approximately spherical double layer with an interior or interior 2 out. In the example shown, GUVs are produced, ie very large liposomes. The preparation takes place in a medium, in which, inter alia, substances 5 such as glycerol, sucrose or dextran dissolved which increases the density of the solution. This solution with the substance 5 is also in the interior 2 the vesicle 1 available.

The vesicles 1 The GUVs are then harvested and placed in a lower density buffer solution (not shown), ie, the buffer solution contains the substances 5 not or in a lower concentration. The buffer solution also contains one or more fusogenic substances, for example calcium chloride. The buffer solution with the vesicles 1 is then given a (not shown) centrifuge container, at the bottom of a structured Messchip 100 is arranged as a solid support. The Messchip 100 has depressions 101 which later serve as measuring chambers. The aperture of the measuring chambers 101 can be in the range of micrometers to a few nanometers as required. The centrifuge container is hung in or attached to a swinging bucket rotor (not shown) and the centrifuge is turned on.

As 1b shows are inside 2 the vesicle 1 continue the substances 5 because the bilayer of vesicles 1 for the substances 5 is not permeable or at least for the duration of the centrifugation, the substance 5 not appreciably let through. By this loading of the substance 5 is the density of the vesicles 1 increased in relation to the surrounding buffer solution. By centrifuging is on the vesicles 1 therefore a strong force 6 exerted orthogonal to the surface of the measuring chip 100 affects and the vesicles 1 accelerated in his direction.

1c shows that the vesicles 1 after a certain centrifugation time on the measuring chip 100 impact, collapse, lose their spherical structure and finally open from above.

1d shows that the vesicles 1 fully open on further centrifugation and on the measuring chip 100 a planar lipid bilayer 7 have trained. The measuring chamber shown 101 is from the planar lipid bilayer 7 completely covered and sealed at the top.

The 1e and 1f show how in the in 1d illustrated planar lipid bilayer 7 membrane proteins 8th be incorporated by fusion. For this purpose, first vesicles 10 which are smaller than GUVs, called LUVs (see above). The LUVs were made into membrane proteins during production 8th reconstituted so that they are therefore proteoliposomes. As with the in 1a to 1b shown vesicles 1 are also the smaller vesicles 10 in her interior 2 with substances 5 filled or loaded, for example with sucrose, the density of the vesicles 10 relative to the surrounding (not shown) buffer solution in which the vesicles 10 be centrifuged. The buffer solution also contains one or more fusogenic substances, for example calcium chloride.

The vesicles 10 then go in the direction 6 of the measuring chip 100 centrifuged, where they are with the on the Messchip 100 arranged planar lipid bilayer 7 merge. By fusion, the membrane proteins arrange 8th from the vesicles 10 in the lipid bilayer 7 at.

The Messchip 100 is then removed from the centrifuge and can be used for measurements. Is it the membrane proteins 8th for example, a channel protein containing a substrate (not shown) via the lipid bilayer 7 transported, then the substrate accumulates in the measuring chamber 101 and can there by measurements, such as fluorescence imaging, be detected quantitatively and time resolved.

The 2a and 2 B illustrate an example of the method by having a planar lipid bilayer 7 with membrane proteins 8th can be produced in only one centrifugation step. The process essentially corresponds to that in the 1a to 1d illustrated and described steps. So are inside 2 the vesicle 1' substances 5 , for example dextran, which controls the density of the vesicles 1' increase in relation to the surrounding buffer solution.

The difference is that as a vesicle 1 GUVs are used in the membrane proteins during or after manufacture 8th were reconstituted. So these are proteoliposomes. Instead of GUVs, however, smaller proteoliposomes can also be used, for example LUVs, as described in US Pat 1e is shown. Centrifugation causes the vesicles to hit 1' on the Messchip 100 on and form on the Messchip 100 a planar lipid bilayer 7 with the reconstituted membrane proteins 8th ,

The Messchip 100 can then be removed and the properties of the lipid membrane and the membrane proteins are directly examined by the above-mentioned measuring methods.

Because of its speed and simplicity, the method described is particularly suitable for high throughput screenings and measurements.

LIST OF REFERENCE NUMBERS

1

 Great vesicles

2

 inner space

3

 lipid heads

4

 lipid tails

5

 substance

6

 force vector

7

 Lipid bilayer

8th

 membrane protein

10

 Small vesicles

100

 measuring chip

101

 measuring chamber

Claims (10)

Process for the preparation of planar lipid bilayers ( 7 ) on a solid support ( 100 ), which comprises the following steps: a) one or more vesicles ( 1 . 10 ) are prepared or provided, b) the solid support ( 100 ) is placed in a suitable centrifuge container in a centrifuge, c) the vesicles ( 1 . 10 ) are added together with a solution in the centrifuge container, whereby the vesicles ( 1 . 10 ) have a higher density than the density of the solution, d) the centrifuge container with the vesicles ( 1 . 10 ) and the solution is centrifuged until the vesicles ( 1 . 10 ) on the support ( 100 ) a lipid bilayer ( 7 ) train. Method for fusion of vesicles ( 1 . 10 ) with planar lipid bilayers ( 7 ) on a solid support ( 100 ), which comprises the following steps: a) one or more vesicles ( 1 . 10 ) are prepared or provided, b) the solid support ( 100 ) with at least one planar lipid bilayer ( 7 ) is placed in a suitable centrifuge container in a centrifuge, c) the vesicles ( 1 . 10 ) are added together with a solution in the centrifuge container, whereby the vesicles ( 1 . 10 ) have a higher density than the density of the solution, d) the centrifuge container with the vesicles ( 1 . 10 ) and the solution is centrifuged until the vesicles ( 1 . 10 ) with the lipid bilayer ( 7 ) on the support ( 100 ) merge. Method according to claim 2, characterized in that the planar lipid bilayers ( 7 ) on a solid support ( 100 ) was prepared according to the method of claim 1. Method according to one of the preceding claims, characterized in that the vesicles ( 1 . 10 ) in its interior ( 2 ) have a solution whose density is higher than the density of the solution added to the centrifuge container. Method according to the preceding claim, characterized in that the solution comprises one or more substances ( 5 ), which increase the density, preferably glycerol, dextran, sucrose, other sugars, salts, or nanoparticles. Method according to one of the preceding claims, characterized in that the vesicles ( 1 . 10 ) Liposomes or proteoliposomes ( 10 ' ), in particular GUVs, LUVs, SUVs or virus particles, or that the vesicles ( 1 . 10 ) are formed from natural, biological membranes. Method according to one of the preceding claims, characterized in that the solution introduced into the centrifuge container has one or more fusogenic substances, in particular divalent ions such as calcium chloride, pore formers such as nystatin-cholesterol, complexing agents such as EDTA or SNARE proteins. Method according to one of the preceding claims, characterized in that a centrifuge is used with a swing-bucket rotor. Method according to one of the preceding claims, characterized in that the solid support is a glass plate, preferably a cover glass for microscopy, or a micro- or nanostructured Messchip ( 100 ). The supports may have a functionalized surface that facilitates surface fusion. Several carriers can be arranged in multiwell formats, eg in 96-well formats, and placed in the centrifuge at the same time. Method according to one of the preceding claims, characterized in that the lipid bilayers produced ( 7 ) are measured by scanning probe microscopy, impedance spectroscopy, fluorescence imaging, quartz crystal microbalances (QCM), surface plasmon resonance spectroscopy or electrophysiologically.

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