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WO2007031152A1 - Procede et dispositif pour produire des membranes artificielles spheriques ou tubulaires - Google Patents

Procede et dispositif pour produire des membranes artificielles spheriques ou tubulaires Download PDF

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Publication number
WO2007031152A1
WO2007031152A1 PCT/EP2006/007585 EP2006007585W WO2007031152A1 WO 2007031152 A1 WO2007031152 A1 WO 2007031152A1 EP 2006007585 W EP2006007585 W EP 2006007585W WO 2007031152 A1 WO2007031152 A1 WO 2007031152A1
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WO
WIPO (PCT)
Prior art keywords
channels
intermediate layer
film
pressure difference
lipid
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2006/007585
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German (de)
English (en)
Inventor
Petra Dittrich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leibniz Institut fuer Analytische Wissenschaften ISAS eV
Original Assignee
Leibniz Institut fuer Analytische Wissenschaften ISAS eV
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 Leibniz Institut fuer Analytische Wissenschaften ISAS eV filed Critical Leibniz Institut fuer Analytische Wissenschaften ISAS eV
Publication of WO2007031152A1 publication Critical patent/WO2007031152A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases

Definitions

  • the invention relates to a process for the production of spherical or tubular artificial membranes from self-assembling film-forming molecules, in particular from lipids, in which the film-forming molecules are contacted in layers from two sides with a solution, in particular an aqueous solution, and a device for Implementation of this procedure.
  • membranes One of the tasks that membranes have to fulfill in nature is the formation of compartments that delimit the enclosed areas from the environment. Thereby, e.g. high osmolarity (high ion concentration) is maintained within the cell against low osmolarity outside the cell. Only special processes can be used to exchange substances. Membranes also have cohesiveness and stability, for example, to provide a transport pathway for nerve cells in different cell components.
  • lipid membranes are also of great importance to biotechnology.
  • Various methods have been developed to produce lipid vesicles. As early as the mid-sixties of the last century, it was observed that lipid films swell in aqueous solution, ie, water penetrates into the lipid film and forms vesicles of various sizes. By applying electric fields or As the temperature increased, the process of vesicle formation could be accelerated. A spontaneous formation of very small vesicles (100 nm to 250 nm) could be achieved on a microchip. In this case, an organic phase containing lipid in dissolved form was hydrodynamically focused by means of two laterally inflowing aqueous solutions. At the interface between the organic phase and the aqueous solution, the vesicles are formed.
  • the preparation methods are based on the property of lipids or other self-assembling film-forming molecules to spontaneously assemble into an ordered, single or multi-layered (so-called unilamellar or multilamellar) membrane. There will be no chemical (Covalent) bonds formed, but there are only interactions between the lipids, so that the structures can be easily changed under appropriate conditions in their shape or composition.
  • membrane hoses were drawn from spherical vesicles, e.g. by means of a pipette, in which there is a slight negative pressure.
  • the hoses thus produced usually have a diameter in the range of 20 to 200 nm, the maximum length which has been described is a few hundred micrometers.
  • the membrane tubes are only stable as long as they are attached at both ends, i. they form back when the vacuum of the pipette is released.
  • the pipette method has also been used to create networks of nanochannels that connect different vesicles or vesicles with biological cells. However, since these channels are only metastable, arbitrary arrangements of networks are not possible. It has also been observed that lipid tubing in fluid systems forms from cell membranes or vesicles, which regress as the flow is stopped.
  • the object of the invention is to provide a solution with which such artificial membranes in spherical or tubular form reproducible, stable and largely automated can be produced.
  • This object is achieved by a method of the type described in the present invention that the film-forming molecules on a two-channel microchip separating, microporous separating intermediate layer is applied and the two channels are each filled with an aqueous solution and that then between the two Channels a pressure difference is generated.
  • a method of the type described in the present invention that the film-forming molecules on a two-channel microchip separating, microporous separating intermediate layer is applied and the two channels are each filled with an aqueous solution and that then between the two Channels a pressure difference is generated.
  • spherical, ie spherical structures and also tubular structures of self-assembling film-forming molecules, in particular of lipids, with diameters up to a few micrometers can be produced on a microchip.
  • the pore-containing intermediate layer between the two channels of the microchip is coated with a film-like layer of the film-forming molecules, ie, such a film is applied to the intermediate layer.
  • the film-forming molecules are preferably contained in an organic solution as a solvent.
  • the two channels are filled with water or an aqueous solution.
  • the forming membrane structures are then forced through the pores in the direction of the side facing away from the film, so to speak, extruded, depending on the dimensions of the micropores and the pressure difference or the flow velocity in the channels reproducible three-dimensional structures artificial membranes can be generated.
  • These membranes can be used as microreactors or transport channels for biotechnological or pharmaceutical applications.
  • the aqueous solution is sucked out of a channel. If the pressure difference is small, spherical and cylindrical, tori-shaped compartments (tori) are preferably formed. If no shear forces act on the back of the micropores, the membranes or vesicles formed adhere there.
  • Producing a high pressure difference (by sucking on the side facing away from the film) produces tubes that can reach a length of more than 1.5 cm. These hoses are stable even when the pressure difference is stopped ie they no longer change their diameter. They are flexible against movement, ie they can be moved or rotated in any direction.
  • the diameter of the lipid tubes is (presumably) determined by the diameter of the pores in the thin intermediate layer, which can be varied.
  • the geometry of the microchip can be easily changed, e.g. the design of the channels, the filling holes and the pattern of the pores. Any number of membrane structures can thus be generated simultaneously and arranged as desired.
  • volume flow of the order of 1 to 100 .mu.l / min. aspirated, with larger volumetric flows, i. large pressure differences, tubular membranes arise.
  • volume flows refer to microchips with channels in the order of 100 ⁇ m wide and 30 ⁇ m high.
  • an intermediate layer is used with pores whose diameter is in the order of 2 to 3 microns.
  • aqueous solutions are introduced into the two channels.
  • Organic solutions can also be used.
  • the invention also provides an apparatus for performing the method described above, which has a microchip with two channels, which are separated by a microporous intermediate layer, wherein a channel with a pressure difference between the two channels generating device is connected.
  • This device for generating a pressure difference is preferably designed as a pump, so that the pressure difference is generated by the fact that from the (rear) channel, the aqueous solution is sucked off with the forming membranes.
  • the micropores in the intermediate layer have diameters in the order of a few microns.
  • the channels are between 10 and 100 microns wide and high.
  • the method described above and the device described above enable automated reproducible production of microreactors or micro-tubes of homogeneous size.
  • By separating the areas in front of and behind the thin intermediate layer it is possible to directly fill the membranes or vesicles, or any desired number of substances can be passed through the lipid tubes in a targeted manner, ie. the solution enclosed within the structures may be different than the surrounding solution. No further purification / separation steps are required. Since all processes take place on a microchip and in an aqueous phase, further reaction / manipulation / analysis steps can be carried out directly on the chip.
  • the vesicles can be used as microreactors, eg for carrying out and analyzing biochemical / chemical reactions. Since they can be produced at high speed (presumably up to 100 vesicles per second and per pore) and in always identical size, can they are used as carriers of active ingredients in pharmacy / medicine, as well as for drug development. The range of application can be extended by inserting membrane proteins into the vesicle membrane.
  • the membrane hoses are generated stress-free and not bound to a vesicle. As long as sufficient material is available during production, the diameter of the channels does not become smaller with increasing length or retracts again after the pressure difference has disappeared. It is likely that they can be prepared by suitable methods, e.g. optical busting or micromanipulation can be interconnected so that complex but changeable networks of lipid tubing can be constructed. Both structural types, both spherical and tubular membranes, can further contribute to solving fundamental issues in cell biology (e.g., how to transport substances along a lipid tube, etc.).
  • 1 is a side view of an apparatus for performing the method
  • FIG. 1 a an enlarged detail of FIG. 1,
  • 3 shows a schematic representation of a lipid molecule, a unilamellar lipid membrane and a multi-lamellar lipid membrane
  • 4 is a schematic representation of a spherical vesicle
  • FIG. 5 shows a schematic representation of a lipid tube
  • Fig. 6 is a photograph of a with a
  • FIG. 8 is an illustration of lipid tubes formed on the intermediate layer
  • An apparatus for carrying out the method for producing spherical or tubular artificial membranes from self-assembling film-forming molecules, in particular from lipids, has a microchip, generally designated 1.
  • This microchip 1 has two channels 2, 3, which are contained in two PDMS (silicon) layers of the microchip. Between the two channels 2, 3 an intermediate layer 4 is arranged, which preferably consists of silicon, in particular SiO 2 / Si 3 N 4 .
  • the intermediate layer 4 is provided with micropores in a size of for example 2.5 microns.
  • the microchip 1 On the underside, the microchip 1 has a glass carrier 5 for stabilization.
  • a micropore 6 of the intermediate layer 4 is shown. Furthermore, a forming spherical membrane in the region of the pore 6 is indicated and designated 7.
  • lipid film e.g. 1,2-Dilauroyl-sn-glycero-3-phosphocholine.
  • lipids and amphiphilic molecules can be used, or in other words, self-assembling film-forming molecules.
  • the channels 2 and 3 are filled with water or an aqueous solution, which may also be different aqueous solutions.
  • the lipid film is now pressed through the pores 7. This can be done by positive pressure on the side of the lipid film (the channel 2 side facing), but preferably by negative pressure on the other side of the intermediate layer 4, that is adjacent to the channel 3. For this purpose, preferably at the output of the channel 3 is not shown Pump connected.
  • a pressure difference between the channel 2 and the channel 3 a flow is generated in front of or behind the micropores 6. The magnitude of the pressure difference acting on the micropores 6 influences the resulting membrane structures.
  • tubular membranes are generated, which can reach a length of up to 1.5 cm and more. These tubular membranes are stable, even if no pressure difference exists, ie they no longer change their diameter. They are flexible against movement, ie they can be moved or rotated in any direction.
  • the diameter of the lipid tubes is presumably determined by the diameter of the micropores 6 in the intermediate layer 4, which can be varied within the limits of photolithography.
  • the geometry of the microchip can be easily changed, e.g. the design of the channels 2, 3 of the filling openings and the pattern of the micropores 6.
  • any number of lipid structures can be generated simultaneously and arranged arbitrarily.
  • lipid molecule and lipid membranes forming therefrom are shown schematically by way of example in FIG.
  • a single lipid molecule is shown on the left in FIG. 3, it has a polar head group 8 and two nonpolar hydrocarbon chains 9, 10.
  • FIG. 3 shows a unilamellar lipid membrane 11, as arranged in an aqueous solution, with the polar head groups pointing outwards.
  • FIG. 3 shows a lipid film or a multilamellar lipid membrane 12.
  • FIGS. 4 and 5 are illustrated by the method. end membrane structures.
  • FIG. 4 shows a spherical membrane or, in the case of lipids, a spherical vesicle 13,
  • FIG. 5 shows a tubular membrane 14.
  • FIG. 6 shows the intermediate layer 4 of SiO 2 / Si 3 N 4 with an exemplary width of 100 ⁇ m and pores with a diameter of approximately 2.5 ⁇ m, the intermediate layer being covered with a lipid film which is dyed with a fluorescent dye.
  • FIG. 7 it can be seen that lipid vesicles have formed on the micropores due to the pressure difference between the channels 2, 3 on both sides of the intermediate layer 4.
  • FIG. 8 shows the intermediate layer 4 of SiO 2 / Si 3 N 4 with the individual micropores.
  • FIG. 9 shows the lipid tubes forming in the direction of flow.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Dispersion Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Abstract

L'invention concerne un procédé de production de membranes artificielles sphériques ou tubulaires à partir de molécules filmogènes à auto-organisation, en particulier à partir de lipides, lesdites molécules filmogènes étant mises en contact sous forme de couche sur deux côtés avec une solution, en particulier une solution aqueuse. L'objectif de l'invention est de mettre au point une solution permettant de produire des membranes artificielles sphériques ou tubulaires de ce type de façon reproductible, stable et automatisée. A cet effet, les molécules filmogènes sont appliquées sur une couche intermédiaire présentant des micropores et séparant deux canaux d'une puce électronique, chaque canal est rempli d'une solution aqueuse, puis une différence de pression est établie entre les deux canaux.
PCT/EP2006/007585 2005-09-09 2006-08-01 Procede et dispositif pour produire des membranes artificielles spheriques ou tubulaires Ceased WO2007031152A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200510042884 DE102005042884B3 (de) 2005-09-09 2005-09-09 Verfahren und Vorrichtung zur Herstellung von kugel- oder schlauchförmigen künstlichen Membranen
DE102005042884.3 2005-09-09

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WO2007031152A1 true WO2007031152A1 (fr) 2007-03-22

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085341A1 (fr) * 2000-05-12 2001-11-15 Pyrosequencing Ab Dispositifs microfluidiques
US20020050660A1 (en) * 1990-10-05 2002-05-02 Coe Royden M. Liposome extrusion process
US20030129223A1 (en) * 2000-10-11 2003-07-10 Targesome, Inc. Targeted multivalent macromolecules
US20030150791A1 (en) * 2002-02-13 2003-08-14 Cho Steven T. Micro-fluidic anti-microbial filter

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19640092A1 (de) * 1996-09-28 1998-04-16 Beiersdorf Ag Strukturen mit Lipid-Doppelmembranen, in deren lipophilen Bereich längerkettige Moleküle eintauchen oder durch hydrophobe Wechselwirkungen an solche Moleküle angedockt sind
US20050214163A1 (en) * 2004-03-29 2005-09-29 Takeshi Kinpara Bilayer lipid membrane forming device and bilayer lipid membrane forming method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020050660A1 (en) * 1990-10-05 2002-05-02 Coe Royden M. Liposome extrusion process
WO2001085341A1 (fr) * 2000-05-12 2001-11-15 Pyrosequencing Ab Dispositifs microfluidiques
US20030129223A1 (en) * 2000-10-11 2003-07-10 Targesome, Inc. Targeted multivalent macromolecules
US20030150791A1 (en) * 2002-02-13 2003-08-14 Cho Steven T. Micro-fluidic anti-microbial filter

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ESTES D J ET AL: "Giant liposomes in physiological buffer using electroformation in a flow chamber", BIOCHIMICA ET BIOPHYSICA ACTA. BIOMEMBRANES, AMSTERDAM, NL, vol. 1712, no. 2, 1 July 2005 (2005-07-01), pages 152 - 160, XP004947412, ISSN: 0005-2736 *
LIN ET AL: "Manipulating self-assembled phospholipid microtubes using microfluidic technology", SENSORS AND ACTUATORS B, ELSEVIER SEQUOIA S.A., LAUSANNE, CH, vol. 117, no. 2, 12 October 2006 (2006-10-12), pages 464 - 471, XP005591599, ISSN: 0925-4005 *

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